Novel self-assembling drug amphiphiles and methods for synthesis and use

ABSTRACT

The present invention provides herein the design of monodisperse, amphiphilic anticancer drugs—which are now termed “drug amphiphiles” (DAs)—that can spontaneously associate into discrete, stable supramolecular nanostructures with the potential for self-delivery (no additional carriers are needed). The quantitative drug loading in the resulting nanostructures is ensured by the very nature of the molecular design. The DA is a composition comprising: D-L-PEP; wherein D is 1 to 4 hydrophobic drug molecules which can be the same or different; L is 1 to 4 biodegradable linkers which can be the same or different; and PEP is a peptide that can spontaneously associate into discrete, stable supramolecular nanostructures. In an alternate embodiment, the DA composition also comprises a targeting ligand (T). Methods of making DA molecules, as well as their use in treatment of disease are also provided.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/044,329, filed Oct. 2, 2013, which issued as U.S. Pat. No.9,180,203, and which claims the benefit of U.S. Provisional PatentApplication No. 61/717,447, filed on Oct. 23, 2012, the content of eachof the aforementioned applications is herein incorporated by referencein their entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 2, 2013, isnamed P12082-02_ST25.txt, and is 2,499 bytes in size.

BACKGROUND OF THE INVENTION

The creation of vehicles for the effective delivery of hydrophobicanticancer drugs to tumor sites has garnered major attention in cancerchemotherapies for several decades. A successful strategy promisesimmense benefits to cancer sufferers through both the reduction ofside-effects and a greater treatment efficacy. Current approaches focuson the use of nanocarriers, whereby the drug's pharmacokineticproperties and biodistribution profiles are manipulated by encapsulationwithin liposomes, polymeric nanoparticles or micelles, or by conjugationto hydrophilic polymers or inorganic nanomaterials. While these methodscan be effective, there are concerns regarding the short-term andlong-term toxicities arising from the synthetic nanomaterials other thanthe drug being delivered. This often leads to exhaustive preclinicalevaluation and thus represents a difficult hurdle for the drug'stranslation into clinical use. Furthermore, there are inherentdifficulties in achieving a quantitative and high drug loading percarrier, and the drug loading capacity varies depending not only on thecarrier's properties but also on the type of drugs to be encapsulated orconjugated. Polydispersity, both in terms of polymer length and theamount of drug loaded or conjugated, is a critical issue susceptible tosignificant batch-to-batch variability. On the other hand, smallmolecule prodrugs are monodisperse but can be subject to rapid clearanceand premature degradation

Many drugs, including chemotherapeutic drugs for cancer are well knownfor having low water solubility, for example, camptothecin andpaclitaxel. To circumvent this, two strategies have beenadopted—chemical modification of the drug to increase solubility or theuse of a delivery vehicle. Camptothecin, a DNA-Topoisomerase I inhibitorthat prevents DNA re-ligation during transcription and ultimately causescell apoptosis, is not currently used in clinical cancer chemotherapydue to its very low water solubility and toxic side effects; however,its more soluble derivatives have successfully made the transition intoclinical use, such as Topetecan (HYCAMTIN, GlaxoSmithKline) andIrinotecan (CAMPTOSAR, Pfizer and CAMPTO, Yakult Honsha). Thesederivatives still cause significant side-effects due to non-selectivemodes of action, and would benefit from an improved delivery strategy.Paclitaxel, a mitotic inhibitor that stabilizes microtubules, preventingcell division and inducing apoptosis, has for many years beenadministered intravenously as a solution in Chremophor EL (CrEL), aformulation known as (TAXOL, Bristol-Myers Squibb). The Chremophor ELsolvent, however, causes side-effects of its own in addition to thosedue to paclitaxel and alternatives are highly desired. In 2005, aninjectable formulation of paclitaxel in which the drug is bound to theprotein albumin was approved for use by the FDA. Known as (ABRAXANE,Celgene), this mode of delivery represents the first nanoparticlealbumin bound (nab) technology platform. While the carrier causes littleto no side-effects, those due to the paclitaxel are still present.

As such, there still exists a need for improved compositions and methodsfor solubilizing drug compounds that have low water solubility withoutinducing unwanted secondary biological effects due to the solubilizationmethods.

SUMMARY OF THE INVENTION

The present invention provides herein the design of monodisperse,amphiphilic anticancer prodrugs—which are now termed “drug amphiphiles”(DAs)—that can spontaneously associate into discrete, stablesupramolecular nanostructures with the potential for self-delivery (noadditional carriers are needed). The very nature of the molecular designensures that a fixed and tunable drug loading can be achieved. Assemblyof these DAs provides a basis for increasing the drug solubility andstability to non-specific degradation, and for improving specifictargeting to tumor cells, with concomitant reduction in systemictoxicity towards healthy tissues and improved treatment efficacy.

In order to imbue these properties upon an anticancer drug, ahydrophilic peptide is conjugated to the drug via a biodegradablelinker. The hydrophilic peptide increases the aqueous solubility of thedrug and can promote the formation of well-defined nanostructurearchitectures including, but not limited to, cylindrical or sphericalmicelles, hollow nanotubes, toroids, discs and vesicles, throughpreferred secondary structure formation, e.g. beta sheet, alpha helix,poly proline type-II helix, beta turn. The peptide may also includemoieties that will allow the assembled nanostructure to preferentiallyaccumulate at tumor sites using established targeting strategies such asfolate ligands or integrin-binding peptides (RGDS for example), and/orthat can improve overall pharmokinetics, e.g. pegylation. Thebiodegradable linker can be sensitive to cleavage by a number oftumor-relevant stimuli including, but not limited to, reducing agents(glutathione, cysteine, etc), proteolytic enzymes (Cathepsins, MatrixMetalloproteases, etc) and low pH (endosomal/lysosomal pH). Thedrug-linker can be conjugated to the hydrophilic peptide via establishedprotein conjugation methodologies including, but not limited to,disulfide formation via reaction of cysteine-thiol with an activatedthiol, thioether formation via reaction of cysteine-thiol with amaleimide, triazole formation via copper-assisted azide-alkynecycloaddition (CuAAC) and other “Click” reactions.

In one embodiment of the invention the Tau-protein derived sequenceGVQIVYKK (SEQ ID NO: 1) was conjugated to two known chemotherapeuticagents, camptothecin (CPT) and paclitaxel (PXL), which are models forlow water soluble chemotherapeutic agents, and which resulted in severalbenefits in antitumor treatment therapeutics. First, the DA was composedof hydrophobic CPT or PXL and hydrophilic Tau peptide, which enables theconjugate DA to form nanostructures for the delivery process.Furthermore, the preference of the Tau peptide for beta sheet formationpromotes the formation of nanofiber-like structures. Second, byconjugating the drugs to a peptide sequence instead of encapsulating thedrug into polymers, a highly improved drug loading was observed. Third,a biodegradable disulfide bond linker (e.g., disulfanylbutanoate (buSS))was utilized, which can be degraded by glutathione and other reducingagents within tumor cells to allow controlled release during the drugdelivery process. Fourth, the formation of nanostructures effectivelyshields the drug and linker moieties from the external environment, onlydisplaying significant release of the active drug when in the monomericform or when exposed to glutathione or other reducing agents. Thisproperty allows for increased extracellular stability, with cleavageoccurring upon cellular internalization.

In accordance with an embodiment, the present invention provides a DAcomposition comprising: D-L-PEP; wherein D is 1 to 4 hydrophobic drugmolecules; L is 1 to 4 biodegradable linkers; and PEP is a hydrophilicpeptide that can promote the formation of specific nanostructurearchitectures.

In accordance with another embodiment, the present invention provides acomposition comprising 1 to 4 hydrophobic chemotherapeutic moleculeslinked via 1 to 4 (disulfanylbutanoate) (buSS) linking molecules tocysteine-modified analogues of the Tau-protein derived peptide GVQIVYKK(SEQ ID NO: 1).

In accordance with a further embodiment, the present invention providesa method for making the compositions described above comprising reactionof the 4-(pyridin-2-yldisulfanyl)butanoate-functionalized CPT or PXLwith a cysteine-functionalized hydrophilic peptide to furnish thedescribed DA conjugates.

In accordance with an embodiment, the present invention provides a DAcomposition comprising: D-L-PEP-T; wherein D is 1 to 4 hydrophobic drugmolecules; L is 1 to 4 biodegradable linkers; and PEP is a hydrophilicpeptide that can promote the formation of specific nanostructurearchitectures, and T is a targeting ligand.

In accordance with yet another embodiment, the present inventionprovides a method of treating a disease in a mammal comprisingadministering to the mammal a therapeutically effective amount of thecompositions described above, sufficient to slow, stop or reverse thedisease in the mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D illustrates an embodiment of the invention, including thestructures of the drug amphiphiles (DAs) and control molecules. a). Theself-assembled nanostructures contain the same drug fraction as theindividual DA. b). The three key component parts of a drug amphiphilestudied in this paper: the hydrophobic drug CPT, the Tau β-sheet formingpeptide, and the buSS biodegradable linker. c). The synthesized CPT DAswith quantitative drug loadings of 23%, 31% and 38%. d. The twosynthesized control molecules.

FIGS. 2A-2H shows the TEM characterization of the drug amphiphiles. TEM(a) and cryo-TEM (b) images of long filaments of widths 6.7±1 nm formedby mCPT-buSS-Tau in water and a self-supporting gel formed at 5 mM in1×PBS (inset). TEM (c) and cryo-TEM (d) images of shorter filaments ofwidths 7.2±1.4 nm formed by dCPT-buSS-Tau in water. TEM (e) and cryo-TEM(f) images of nanotubes of widths 9.5±1 nm formed by qCPT-buSS-Tau inwater. The cryo-TEM (f) resolution is insufficient to show the tubularnature. (g) High resolution TEM image of the tubular morphology formedby qCPT-buSS-Tau. The circular shape of the terminal ends (marked withwhite arrows) confirms the tubular structures. (h) TEM images of longnanotubes formed by qCPT-buSS-Sup35. (i) Schematic illustration of theproposed nanotube morphology. TEM samples for images of (a), (c), (e),(g) and (h) were stained with 2% uranyl acetate aqueous solution toenhance the image contrast. Solution concentrations: 50 μM for (a), (c),(e), (g) and (h); 1 mM for (b) and (d); 100 μM for (f).

FIGS. 3A-3F shows the spectroscopic analysis and release study of thedrug amphiphiles. Circular dichroism (CD, solid line) and UV-Vis (dashedline) spectroscopic analysis of 1 μM mCPT-buSS-Tau (a), 1 μMdCPT-buSS-Tau (b) and 1 μM qCPT-buSS-Tau (c) in 10 mM sodium phosphate.Mean residue ellipticity values are given in10³·deg·cm²·dmol⁻¹·residue⁻¹. Release study of 2 μM DA and controlmolecules in the presence and absence of 10 mM glutathione (GSH) in 10mM sodium phosphate at 37° C. (d). Comparison of mCPT-buSS-Tau releasekinetics at 2 μM and 20 μM (e). Release experiments were performed intriplicate and values are given as mean±s.d. (Key: mono=mCPT-buSS-Tau,di=dCPT-buSS-Tau, quad=qCPT-buSS-Tau, maleimide=mCPT-mal-Tau). Schematicillustration of the proposed release mechanism showing the effect ofself-assembly on the susceptibility of the DAs to degradation (f).

FIGS. 4A, 4B, 4D shows the CD spectra for mCPT-buSS-Tau (a),dCPT-buSS-Tau (b) and qCPT-buSS-Tau (d) at pH 3.5 (1 mM HCl) and inDMSO, showing persistence of the nanostructures under more acidicconditions and their existence as single molecules in DMSO. BothmCPT-buSS-Tau and dCPT-buSS-Tau show little to no signal in DMSO,whereas qCPT-buSS-Tau shows a relatively stronger signal. All solutionswere 1 μM, with the exception of qCPT-buSS-Tau which was 500 nM in DMSO.

FIGS. 5A-5C depicts the fluorometric determination of the CMC values formCPT-buSS-Tau (a), dCPT-buSS-Tau (b) and qCPT-buSS-Tau (c) in 10 mMsodium phosphate.

FIG. 6 shows the fluorescence spectra of 1 μM solutions ofmCPT-buSS-Tau, dCPT-buSS-Tau and qCPT-buSS-Tau in 10 mM sodium phosphate(λ_(em)=350 nm).

FIGS. 7A-7B shows an in vitro dose-response relationship study of the DAmolecules against human MCF-7 breast cancer (a) and rat 9L gliosarcoma(b) cells. All cancer cells were incubated with the appropriate DAmolecules for 48 hours and cell viability was determined by SRB assay.Cytotoxicity experiments were performed in triplicate and values aregiven as mean±s.d. (n=3). Key: see FIG. 3

FIGS. 8A-8F shows an in vitro dose-response relationship study of the DAmolecules against rat F98L gliosarcoma cells (a), and dCPT-buSS-Tauagainst murine ID-8 ovarian cancer (b), human NCI-H82—small cell lungcancer (c) and human TC-1 cervical cancer (d) cells human A459 non-smallcell lung cancer (e) and human MDA-MB-231 breast cancer (f). All cancercells were incubated with the appropriate DA molecules for 48 hours andcell viability was determined by SRB assay (n.d.=not determined).

FIGS. 9A-9D illustrates the solid-phase synthesis of thecysteine-functionalized Tau precursor peptides. Fmoc-GVQIVYKK-Rink wascreated using an automated peptide synthesizer and further modified bymanual synthesis techniques. Reaction conditions: (a) (i) 20%4-methylpiperidine in DMF, (ii). Fmoc-Lys(Fmoc)-OH, HATU, DIEA (4:3.98:6per amine); (b) (i). 20% 4-methylpiperidine in DMF, (ii)Fmoc-Cys(Trt)-OH, HATU, DIEA (4:3.98:6 per amine), (iii) 20%4-methylpiperidine in DMF, (iv) 20% acetic anhydride in DMF, DIEA; (c)TFA, TIS, H2O (95:2.5:2.5); (d) TFA, TIS, H₂O, EDT (90:5:2.5:2.5).

FIG. 10 illustrates the scheme for the synthesis of the CPT-linkermolecules, CPT-buSS-Pyr and CPT-mal.

FIGS. 11A-11B (A) ¹H NMR (CDCl₃, 400 MHz, 298K) and (B)¹³C NMR (CDCl₃,100 MHz, 298K) of CPT-buSS-Pyr.

FIGS. 12A-12B (A) ¹H NMR (CDCl₃, 300 MHz, 298K) and (B)¹³C NMR (CDCl₃,100 MHz, 298K) of CPT-Mal.

FIGS. 13A-13B (A) depicts the synthesis and (B) chemical structures ofthe drug amphiphiles of the present invention.

FIGS. 14A-14B depicts RP-HPLC (A) and MALDI-Tof MS (B) characterizationof mCPT-buSS-Tau.

FIGS. 15A-15B shows RP-HPLC (A) and MALDI-Tof MS (B) characterization ofdCPT-buSS-Tau. In-source fragmentation was observed corresponding to theloss of one CPT moiety (indicated by *).

FIGS. 16A-16B is the RP-HPLC (A) and MALDI-Tof MS (B) characterizationof qCPT-buSS-Tau. In-source fragmentation was observed corresponding tothe loss of one CPT moiety (indicated by *). Higher laser power resultedin the loss of further CPT fragments.

FIG. 17 is the general scheme for the synthesis of the control peptides.

FIG. 18 depicts RP-HPLC (left) and MALDI-Tof MS (right) characterizationof mCPT-mal-Tau.

FIGS. 19A-19B shows RP-HPLC (A) and MALDI-Tof MS (B) characterization ofC₈-Tau.

FIG. 20 is a CD spectrum of 10 μM qCPTbuSS-Sup35 in 10 mM sodiumphosphate.

FIGS. 21A-21B is the RP-HPLC (A) and ESI MS (B) characterization ofqCPT-buSS-Sup35.

FIG. 22 illustrates the molecular structure of mCPT-etcSS-Pyr

FIG. 23 depicts the synthesis of the carbonate-based linkerCPT-etcSS-Pyr.

FIG. 24 shows the mechanism of the glutathione-induced release of CPTfrom mCPT-etcSS-Tau

FIGS. 25A-25B shows the self-assembly characterization ofmCPT-etcSS-Tau. Representative TEM image of a 100 μM aqueous solution of4 (a), showing the filamentous nanostructures formed by this conjugate.Circular dichroism (CD) spectrum of a 100 μM aqueous solution ofmCPT-etcSS-Pyr (b), indicating the β-sheet secondary structure adoptedby this drug amphiphile and the presence of signals due to the CPTmolecules being in a chiral environment.

FIGS. 26A-26C depicts the HPLC chromatograms of a 50 μM solution ofmCPT-etcSS-Pyr in 10 mM sodium phosphate with 10 mM GSH at roomtemperature after 50 min (solid trace) and overnight incubation (dottedtrace) (a). The traces indicate that CPT is effectively released withlittle to no formation of any isolable intermediary species; kineticstudy of of mCPT-etcSS-Tau (50 μM) degradation in the presence andabsence of 10 mM GSH (b); cytotoxicity study comparing free CPT,mCPT-buSS-Tau (1) and mCPT-etcSS-Tau (4) against the MCF-7 breast cancercell line (c). Cell viability was determined by SRB assay and data arepresented as mean±s.d. (n=3). The calculated IC50 values are given inthe figure legend.

FIG. 27 depicts the (top)¹H NMR (400 MHz, CDCl₃) and (bottom)¹³C NMR(100 MHz, CDCl₃) of CPT-etcSS-Pyr.

FIGS. 28A-28B depicts the RP-HPLC (a) and MALDI-Tof MS (b)characterization of 4. In-source fragmentation was observedcorresponding to the loss of one CPT-O—C(═O)— moiety (indicated by *).

FIG. 29 is a schematic illustration of the structure of PXL-buSS-Tau andthe nanofiber conformation it takes in aqueous solutions.

FIGS. 30A-30D shows TEM images of PXL-buSS-Tau. FIGS. 30 a) and b): 200μM in water. FIGS. 30 c) and d): 10 μM in water.

FIGS. 31A-31C shows the CMC value and CD spectrum of PXL-buSS-Tau. FIG.31 a) maximum wavelength showing that the CMC value lies between 10 μMand 50 μM. FIG. 31 b) CD spectrum of conjugate solution at 5 μM inwater. FIG. 31 c) CD spectrum of conjugate solution at 100 μM in water.

FIG. 32 depicts the PXL-buSS-Tau release curve in PBS buffer under 37°C.

FIGS. 33A-33C shows an in vitro dose-response relationship study ofPXL-buSS-Tau against human MCF-7 breast cancer (a) human A549 non-smallcell lung cancer (b) and human PC-3 FLU prostate cancer (c) cells. Allcancer cells were incubated with the appropriate DA molecules for 48hours and cell viability was determined by SRB assay. Cytotoxicityexperiments were performed in triplicate and values are given asmean±s.d. (n=3).

FIG. 34 is an RP-HPLC analysis of PXL-buSS-Pyr.

FIG. 35 is an ESI-MS characterization of PXL-buSS-Pyr.

FIG. 36 shows the RP-HPLC analysis of PXL-buSS-Tau.

FIG. 37 is an ESI-MS characterization of PXL-buSS-Tau.

FIG. 38 depicts the synthesis of a hetero-dual drug amphiphile (DA)embodiment, CPT-PXL-Sup35, and homo-dual DAs, dCPT-Sup35 and dPXL-Sup35,from the reaction of dCys-Sup35 with a 1:1 mixture of activateddisulfide drugs, CPT-buSS-Pyr and PXL-buSSPyr.

FIGS. 39A-39B shows the reversed-phase HPLC analysis during thesynthesis of a dual drug amphiphile embodiment. Preparative HPLCchromatograms of the reaction of dCys-Sup35 with (39 a) 1 equivalent ofa 1:1 mixture of CPT-buSS-Pyr and PXL-buSS-Pyr after overnight reactionand (39 b) 3 equivalents of a 1:1 mixture of CPT-buSS-Pyr andPXL-buSS-Pyr after 5 days reaction. Percentages give the relativeamounts of each species formed, as determined by calibration of theisolated conjugates. The 220 nm traces (dark) show absorptions from bothCPT and PXL, whereas the 370 nm trace (light) shows CPT absorptionsonly.

FIGS. 40A-40F is a self-assembly study of CPT-PXL-Sup35. Representativeransmission electron microscopy (TEM) images of a 100 μM solution ofCPT-PXL-Sup35 in water 1 hour (40 a and b), 24 hours (40 c) and 48 hours(40 d) after dilution of a 1 mM stock solution that had been allowed toage for 2 hours. Cryo-TEM image of a 500 μM solution of CPT-PXL-Sup35,diluted from a 1 mM sample that had been aged for several days prior tosample preparation (40 e). Circular dichroism (CD) spectra of the 100 μMsolution in water that was monitored over 3 days, showing the formationof the β-sheet secondary structure after 24 hours of incubation (40 f).TEM samples were negatively stained with 2 wt % uranyl acetate.

FIGS. 41A-41C shows a series of TEM images showing the effect ofdilution into phosphate-buffered saline (PBS) on the self-assembly ofCPT-PXL-Sup35. Dilution of a 1 mM CPT-PXL-Sup35 solution in H₂O after 2hours aging to give a 100 μM solution in PBS was found to significantlyslow the formation of the twisted fibril morphology, essentiallycapturing the filament structures (41 a). After 24 hours, little changewas seen though twisted fibrils could be observed on occasion (41 b). Incontrast, similar dilution of a 1 mM CPT-PXL-Sup35 solution that hadbeen allowed to age for 8 days gave only the twisted fibril structure,indicating that PBS does not affect the existing nanostructures (41 c).

FIGS. 42A-42B shows the effect of dilution into PBS on the self-assemblyof CPT-PXL-Sup35. (a) Time course study of a 100 μM sample in PBSprepared from a 1 mM solution in water that had been aged for 2 hours,showing the slow evolution of the β-sheet signal. (b) The CD spectra ofa 100 μM sample in PBS prepared from a 1 mM solution in water that hadbeen aged for 8 days, indicating that dilution into PBS does not disruptthe matured nanostructure.

FIGS. 43A-43B depicts the TEM analysis of the self-assemblednanostructures of dCPT-buSS-Sup35 (100 μM in H₂O) (a) and dPXL-Sup35 (1mM in H₂O) (b).

FIG. 44 depicts the Critical aggregation concentration (CAC)determination of CPT-PXL-Sup35 using a Nile red fluorescence method.Data are given as mean±s.d. (n=2).

FIGS. 45A-45B depicts the Drug release study of 50 μM CPT-PXL-Sup35. (45a) Release of CPT and PXL and degradation of CPT-PXL-Sup35 in thepresence or absence of 10 mM GSH. (45 b) HPLC chromatograms showing theGSH-induced release of CPT, PXL and other intermediates fromCPT-PXL-Sup35. All studies were carried out in 10 mM sodium phosphatesolution with or without 10 mM GSH at 37° C. Data are given as mean±s.d.(n=3). Fitted curves are for illustrative purposes only.

FIGS. 46A-46C depicts the cytoxicity study of the synthesized dual drugamphiphiles against PXL-sensitive KB3-1 cervical cancer cells (46 a),PXL-resistant KB-V1 cervical cancer cells (46 b) and a co-culture ofKB-3-1 and KB-V1 cervical cancer cells (46 c). Both cell lines aresensitive to CPT. Cell viability was determined by SRB assay after 48hours incubation with the appropriate drug-containing media. CalculatedIC50 values are given in the figure legends, * indicates the IC50 valueprior to any observed antagonism. Data are given as mean±s.d. (n=3).

FIG. 47 depicts the HPLC (top) and ESI-MS (bottom) characterization ofpurified dCPT-Sup35.

FIG. 48 depicts the HPLC (top) and ESI-MS (bottom) characterization ofpurified CPT-PXL-Sup35.

FIG. 49 depicts the HPLC (top) and ESI-MS (bottom) characterization ofpurified dPXL-Sup35

DETAILED DESCRIPTION OF THE INVENTION

In accordance with an embodiment, the present invention provides a DAcomposition comprising: D-L-PEP; wherein D is 1 to 4 hydrophobic drugmolecules; L is 1 to 4 biodegradable linkers; and PEP is a hydrophilicpeptide that can promote the formation of specific nanostructurearchitectures.

As used herein, the term “hydrophobic drug molecules” roughly describesa heterogeneous group of molecules that exhibit poor solubility in waterbut that are typically, but certainly not always, soluble in variousorganic solvents. Often, the terms slightly soluble (1-10 mg/ml), veryslightly soluble (0.1-1 mg/ml), and practically insoluble (<0.1 mg/ml)are used to categorize such substances. Drugs such as steroids and manyanticancer drugs are important classes of poorly water-soluble drugs;however, their water solubility varies over at least two orders ofmagnitudes. Typically, such molecules require secondary solubilizerssuch as carrier molecules, liposomes, polymers, or macrocyclic moleculessuch as cyclodextrins to help the hydrophobic drug molecules dissolve inaqueous solutions necessary for drug delivery in vivo. Other types ofhydrophobic drugs show even a lower aqueous solubility of only a fewng/ml. Since insufficient solubility commonly accompanies undesiredpharmacokinetic properties, the high-throughput screening of kinetic andthermodynamic solubility as well as the prediction of solubility is ofmajor importance in discovery (lead identification and optimization) anddevelopment.

As used herein, the term “biodegradable linkers” refers to a smallmolecule or peptide fragment that is capable of covalently linking thehydrophobic drug molecule to the hydrophilic peptide in the presentinvention. These covalent linkages must be sufficiently labile to behydrolyzed or cleaved when in the target cell or organ of a subject. Incertain embodiments, the linker bonds are preferably cleaved off in thetarget organ or cell by an enzyme or cellular component that is at ahigher concentration in the target microenvironment than in the body oroutside of the target cell or organ. Examples of such linker moietiesinclude, but are not limited to amides, disulfides, polyamino acids,biopolymers, esters, aldehydes, hydrazones and the like.

In accordance with an embodiment, the biodegradable linkers of thepresent invention include (4-(pyridin-2-yldisulfanyl)butanoate) (buSS).The buSS linker has a disulfide moiety that allows it to be reductivelycleaved primarily intracellularly by glutathione. In particular, theconcentration of glutathione inside tumor cells is 100 to 1000 timeshigher than in the interstitial fluid, thus allowing the compositions ofthe present invention to act as a prodrug and enter the cell intact.Once inside the cell, the reduction of the linker bonds by glutathioneoccurs, and the free hydrophobic drug molecule can act on its target. Itwill be understood by those of ordinary skill in the art that otherlinker moieties can be used where they interact with the hydrophilicpeptide in a similar manner.

As used herein, the term “tau peptide fragment” means a peptide fragmentof the paired helical filament Tau protein. In an embodiment, thepeptide fragment comprises the amino acid sequence GVQIVYKK (SEQ ID NO:1). The tau peptide fragment is hydrophilic and a strong promoter ofbeta sheet formation, enabling the conjugate to adopt fibrousnanostructures in aqueous solutions. This provides a number ofsignificant features to the composition, including (1) highly improveddrug loading due to the fact that from 1 to 4 drug molecules can bebound to each tau peptide fragment; (2) increased solubility of thehydrophobic drugs due to the presence of the hydrophilic peptide; (3)the nanofiber or nanotube structure of the compositions of the presentinvention partially shields the drug and linker from themicroenvironment, allowing the drug to be released from the conjugate ina controlled manner over time or under highly reducing conditions.

It will be understood by those of ordinary skill in the art that otherpeptide fragments which are hydrophilic and which can form a β-sheet orother secondary structure conformations can also be used in thecompositions of the present invention. Examples of hydrophilic peptidesinclude, but are not limited to, NNQQNY (SEQ ID NO: 2) (from the Sup35yeast prion) and derivatives thereof, GRKKRRQRRRPPQ (SEQ ID NO: 3) (fromthe HIV Tat protein) and derivatives thereof, LLKKLLKLLKKLLK (SEQ ID NO:4) (alpha helical peptide) and derivatives thereof, and de novosequences such as those that possess alternate hydrophobic andhydrophilic residues.

In one or more additional embodiments, the PEP portion of the drugamphiphile of the present invention is selected from the followingpeptide sequences: GVQIVYKK (SEQ ID NO: 1); NNQQNY (SEQ ID NO: 2);GRKKRRQRRRPPQ (SEQ ID NO: 3); LLKKLLKLLKKLLK (SEQ ID NO: 4); CGNNQQNYKK(SEQ ID NO 5); CGVQIVYKK (SEQ ID NO: 6); GN₂Q₂NYK₂ (SEQ ID NO: 7);(GN₂Q₂NY) (SEQ ID NO: 8); (VQIVYK) (SEQ ID NO: 9) and Cys₂KGN₂Q₂NYK₂(SEQ ID NO: 10) and derivatives thereof, wherein the derivativescomprise 1 to 10 additional amino acids on either the N-terminal orC-terminal end of PEP.

In accordance with an embodiment, the present invention provides apharmaceutical composition comprising: D-L-PEP; wherein D is 1 to 4hydrophobic drug molecules; L is 1 to 4 biodegradable linkers; PEP is apeptide comprising a fragment of the Tau protein comprising 6 to 19amino acids of the paired helical filament Tau protein; and one or moreadditional therapeutically active compounds and a pharmaceuticallyacceptable carrier.

It will be understood by those of ordinary skill in the art, that insome embodiments, D can represent two or more different hydrophobic drugmolecules. For example, D can include a first drug (D1) and second drug(D2) which can be, for example, chemotherapeutic agents which are notthe same. In other embodiments, D can represent three or four differentdrug molecules (D1, D2, D3, D4) each linked by a biodegradable linker,which can be the same or different, to a PEP portion of the molecule ofthe present invention. Without being limited to any particular example,the pharmaceutical composition of the present invention can be ahetero-dual drug amphiphile comprising a first drug molecule ofcamptothecin (CPT) and a second drug molecule of paclitaxel (PXL) linkedby the same or different linker, for example buSS, to the PEP portion,for example, Sup35.

In accordance with yet another embodiment, the present inventionprovides a method of treating a disease in a subject comprisingadministering to the mammal a therapeutically effective amount of thecompositions described above, sufficient to slow, stop or reverse thedisease in the mammal.

In accordance with an alternative embodiment, the drug amphiphiles ofthe present invention can be made with a targeting ligand (T) to bind aspecific protein, receptor, or peptide, or other small molecule.

In accordance with an embodiment, the present invention provides a DAcomposition comprising: D-L-PEP-T; wherein D is 1 to 4 hydrophobic drugmolecules; L is 1 to 4 biodegradable linkers; and PEP is a hydrophilicpeptide that can promote the formation of specific nanostructurearchitectures, and T is a targeting ligand.

Without being limited to any particular example, targeting ligands canbe incorporated into the molecular design of the DA conjugates using oneof two approaches, dependent upon their nature. The first isincorporation during the synthesis of the peptide (solid phase) and thesecond is incorporation after the peptide has been purified or once theDA has been synthesized (both solution phase). If performed in solution,the chemistry chosen for incorporation would ideally be orthogonal tothat used to conjugate the drug molecules.

Peptide-based ligands including, but not limited to, integrin bindingpeptides such as RGD, RGDS (SEQ ID NO: 13) and similar derivatives,prostate specific membrane antigen (PSMA) ligands, etc, can be directlyintroduced as part of the peptide sequence (PEP), using the same solidphase Fmoc peptide synthesis techniques.

For example, the following listing of peptides, proteins, and otherlarge molecules may also be used, such as interleukins 1 through 18,including mutants and analogues; interferons a, y, hormone releasinghormone (LHRH) and analogues, gonadotropin releasing hormonetransforming growth factor (TGF); fibroblast growth factor (FGF); tumornecrosis factor-α); nerve growth factor (NGF); growth hormone releasingfactor (GHRF), epidermal growth factor (EGF), connective tissueactivated osteogenic factors, fibroblast growth factor homologous factor(FGFHF); hepatocyte growth factor (HGF); insulin growth factor (IGF);invasion inhibiting factor-2 (IIF-2); bone morphogenetic proteins 1-7(BMP 1-7); somatostatin; thymosin-a-y-globulin; superoxide dismutase(SOD); and complement factors, and biologically active analogs,fragments, and derivatives of such factors, for example, growth factors.

Members of the transforming growth factor (TGF) supergene family, whichare multifunctional regulatory proteins, may be used as the targetingligand in the DAs of the present invention. Members of the TGF supergenefamily include the beta transforming growth factors (for example,TGF-131, TGF-132, TGF-133); bone morphogenetic proteins (for example,BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9);heparin-binding growth factors (for example, fibroblast growth factor(FGF), epidermal growth factor (EGF), platelet-derived growth factor(PDGF), insulin-like growth factor (1GF)), (for example, lnhibin A,lnhibin B), growth differentiating factors (for example, GDF-1); andActivins (for example, Activin A, Activin B, Activin AB). Growth factorscan be isolated from native or natural sources, such as from mammaliancells, or can be prepared synthetically, such as by recombinant DNAtechniques or by various chemical processes. In addition, analogs,fragments, or derivatives of these factors can be used, provided thatthey exhibit at least some of the biological activity of the nativemolecule. For example, analogs can be prepared by expression of genesaltered by site-specific mutagenesis or other genetic engineeringtechniques.

Both peptide-based ligands (as described above) and small moleculetargeting ligands, including but not limited to, folate-receptor bindingmolecules such as folate and methotrexate, can be incorporated usingcommon conjugation techniques. These include, but are not limited to,amide bond formation (requiring a lysine, glutamic acid or aspartic acidgroup at the periphery of the peptide, the C-terminal for instance),reaction with a cysteine thiol (thiol-ene reaction, disulfide formation,thioether formation) or through Click reactions such as azide-alkynecycloaddition. These conjugations may require suitable modification ofthe ligand to provide the required functionality, and may be performedon the solid-phase during synthesis of the peptide or in solution beforeor after the drug molecule(s) is attached.

As used herein the term “pharmaceutically active compound” or“therapeutically active compound” means a compound useful for thetreatment or modulation of a disease or condition in a subject sufferingtherefrom. Examples of pharmaceutically active compounds can include anydrugs known in the art for treatment of disease indications. Aparticular example of a pharmaceutically active compound is achemotherapeutic agent.

The term “chemotherapeutic agent” as well as words stemming therefrom,as used herein, generally includes pharmaceutically or therapeuticallyactive compounds that work by interfering with DNA synthesis or functionin cancer cells. Based on their chemical action at a cellular level,chemotherapeutic agents can be classified as cell-cycle specific agents(effective during certain phases of cell cycle) and cell-cyclenonspecific agents (effective during all phases of cell cycle). Withoutbeing limited to any particular example, examples of chemotherapeuticagents can include alkylating agents, angiogenesis inhibitors, aromataseinhibitors, antimetabolites, anthracyclines, antitumor antibiotics,monoclonal antibodies, platinums, topoisomerase inhibitors, and plantalkaloids. Further examples of chemotherapeutic agents includeasparaginase, busulfan, carboplatin, cisplatin, daunorubicin,doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate,paclitaxel, rituximab, vinblastine, vincristine, etc.

It will be understood that any hydrophobic chemotherapeutic agents canbe conjugated to the biodegradable linker as defined in the presentinvention. Examples include camptothecin, paclitaxel, anthracyclines,carboplatin, cisplatin, daunorubicin, doxorubicin, methotrexate,vinblastine, vincristine, etc.

For purposes of the invention, the amount or dose of the compositions ofthe present invention that is administered should be sufficient toeffectively target the cell, or population of cells in vivo, such thatcell apoptosis or death in the target cell or population of cells occursin the subject over a reasonable time frame. The dose will be determinedby the efficacy of the particular pharmaceutical formulation and thelocation of the target population of cells in the subject, as well asthe body weight of the subject to be treated.

An active agent and a biologically active agent are used interchangeablyherein to refer to a chemical or biological compound that induces adesired pharmacological and/or physiological effect, wherein the effectmay be prophylactic or therapeutic. The terms also encompasspharmaceutically acceptable, pharmacologically active derivatives ofthose active agents specifically mentioned herein, including, but notlimited to, salts, esters, amides, prodrugs, active metabolites, analogsand the like. When the terms “active agent” “pharmacologically activeagent” and “drug” are used, then, it is to be understood that theinvention includes the active agent per se, as well as pharmaceuticallyacceptable, pharmacologically active salts, esters, amides, prodrugs,metabolites, analogs etc.

The dose of the compositions of the present invention also will bedetermined by the existence, nature and extent of any adverse sideeffects that might accompany the administration of a particularcomposition. Typically, an attending physician will decide the dosage ofthe pharmaceutical composition with which to treat each individualsubject, taking into consideration a variety of factors, such as age,body weight, general health, diet, sex, compound to be administered,route of administration, and the severity of the condition beingtreated. By way of example, and not intending to limit the invention,the dose of the pharmaceutical compositions of the present invention canbe about 0.001 to about 1000 mg/kg body weight of the subject beingtreated, from about 0.01 to about 100 mg/kg body weight, from about 0.1mg/kg to about 10 mg/kg, and from about 0.5 mg to about 5 mg/kg bodyweight. In another embodiment, the dose of the pharmaceuticalcompositions of the present invention can be at a concentration fromabout 1 nM to about 10,000 nM, preferably from about 10 nM to about5,000 nM, more preferably from about 100 nM to about 500 nM.

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof cancer in a mammal. Furthermore, the treatment or prevention providedby the inventive method can include treatment or prevention of one ormore conditions or symptoms of the disease, e.g., cancer, being treatedor prevented. Also, for purposes herein, “prevention” can encompassdelaying the onset of the disease, or a symptom or condition thereof.

In accordance with an embodiment of the present invention, themedicament for treating a disease in a subject can encompass manydifferent formulations known in the pharmaceutical arts, including, forexample, intravenous and sustained release formulations. With respect tothe inventive methods, the disease can include cancer. Cancer can be anycancer, including any of acute lymphocytic cancer, acute myeloidleukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breastcancer, cancer of the anus, anal canal, or anorectum, cancer of the eye,cancer of the intrahepatic bile duct, cancer of the joints, cancer ofthe neck, gallbladder, or pleura, cancer of the nose, nasal cavity, ormiddle ear, cancer of the oral cavity, cancer of the vulva, chroniclymphocytic leukemia, chronic myeloid cancer, colon cancer, esophagealcancer, cervical cancer, gastrointestinal carcinoid tumor. Hodgkinlymphoma, hypopharynx cancer, kidney cancer, larynx cancer, livercancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma,nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, pancreaticcancer, peritoneum, omentum, and mesentery cancer, pharynx cancer,prostate cancer, rectal cancer, renal cancer (e.g., renal cell carcinoma(RCC)), small intestine cancer, soft tissue cancer, stomach cancer,testicular cancer, thyroid cancer, ureter cancer, and urinary bladdercancer.

In another embodiment, the term “administering” means that at least oneor more pharmaceutical compositions of the present invention areintroduced into a subject, preferably a subject receiving treatment fora disease, and the at least one or more compositions are allowed to comein contact with the one or more disease related cells or population ofcells.

As used herein, the term “treat,” as well as words stemming therefrom,includes diagnostic and preventative as well as disorder remitativetreatment.

As used herein, the term “subject” refers to any mammal, including, butnot limited to, mammals of the order Rodentia, such as mice andhamsters, and mammals of the order Logomorpha, such as rabbits. It ispreferred that the mammals are from the order Carnivora, includingFelines (cats) and Canines (dogs). It is more preferred that the mammalsare from the order Artiodactyla, including Bovines (cows) and Swines(pigs) or of the order Perssodactyla, including Equines (horses). It ismost preferred that the mammals are of the order Primates, Ceboids, orSimoids (monkeys) or of the order Anthropoids (humans and apes). Anespecially preferred mammal is the human.

Further examples of biologically active agents include, withoutlimitation, enzymes, receptor antagonists or agonists, hormones, growthfactors, autogenous bone marrow, antibiotics, antimicrobial agents, andantibodies. The term “biologically active agent” is also intended toencompass various cell types and genes that can be incorporated into thecompositions of the invention.

In certain embodiments, the subject compositions comprise about 1% toabout 75% or more by weight of the total composition, alternativelyabout 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60% or 70%, of a biologicallyactive agent.

Other non-limiting examples of biologically active agents which can beincluded in the drug amphiphile compositions of the present inventioninclude following: adrenergic blocking agents, anabolic agents,androgenic steroids, antacids, anti-asthmatic agents, anti-allergenicmaterials, anti-cholesterolemic and anti-lipid agents, anti-cholinergicsand sympathomimetics, anti-coagulants, anti-convulsants, anti-diarrheal,anti-emetics, anti-hypertensive agents, anti-infective agents,anti-inflammatory agents such as steroids, non-steroidalanti-inflammatory agents, anti-malarials, anti-manic agents,anti-nauseants, anti-neoplastic agents, anti-obesity agents,anti-parkinsonian agents, anti-pyretic and analgesic agents,anti-spasmodic agents, anti-thrombotic agents, anti-uricemic agents,anti-anginal agents, antihistamines, anti-tussives, appetitesuppressants, benzophenanthridine alkaloids, biologicals, cardioactiveagents, cerebral dilators, coronary dilators, decongestants, diuretics,diagnostic agents, erythropoietic agents, estrogens, expectorants,gastrointestinal sedatives, agents, hyperglycemic agents, hypnotics,hypoglycemic agents, ion exchange resins, laxatives, mineralsupplements, mitotics, mucolytic agents, growth factors, neuromusculardrugs, nutritional substances, peripheral vasodilators, progestationalagents, prostaglandins, psychic energizers, psychotropics, sedatives,stimulants, thyroid and anti-thyroid agents, tranquilizers, uterinerelaxants, vitamins, antigenic materials, and prodrugs.

Specific examples of useful biologically active agents the abovecategories include: (a) anti-neoplastics such as androgen inhibitors,antimetabolites, cytotoxic agents, and immunomodulators.

The “therapeutically effective amount” of the pharmaceuticalcompositions to be administered will be governed by such considerations,and can be the minimum amount necessary to prevent, ameliorate or treata disorder of interest. As used herein, the term “effective amount” isan equivalent phrase refers to the amount of a therapy (e.g., aprophylactic or therapeutic agent), which is sufficient to reduce theseverity and/or duration of a disease, ameliorate one or more symptomsthereof, prevent the advancement of a disease or cause regression of adisease, or which is sufficient to result in the prevention of thedevelopment, recurrence, onset, or progression of a disease or one ormore symptoms thereof, or enhance or improve the prophylactic and/ortherapeutic effect(s) of another therapy (e.g., another therapeuticagent) useful for treating a disease, such as cancer.

In accordance with another embodiment, the present invention providesmethods of treating cancer in a subject comprising administering to themammal a therapeutically effective amount of the composition of thepresent invention sufficient to slow, stop or reverse the cancer in thesubject.

In accordance with an embodiment, the present invention provides apharmaceutical composition comprising a therapeutically effective amountof the compositions described herein, for use in a medicament,preferably for use in treating a proliferative disease in a subject.

In accordance with a further embodiment, the present invention providesa pharmaceutical composition comprising a therapeutically effectiveamount of the compositions described herein, for use in a medicament,preferably for use in treating a tumor in a subject sufficient to slow,stop or reverse the growth of the tumor in the subject.

In accordance with still another embodiment, the present inventionprovides pharmaceutical composition comprising a therapeuticallyeffective amount of the compositions described herein, for use in amedicament, preferably for use in treating cancer in a subjectsufficient to slow, stop or reverse the cancer in the subject.

In accordance with a further embodiment, the present invention providesa method for making the compositions described above comprising: a)dissolving the chemotherapeutic agent in a mixture comprisingdichloromethane and dimethylaminopyridine; b) adding to the solution ofa sufficient amount of 4-(pyridin-2-yldisulfanyl)butanoic acid anddiisopropylcarbodiimide and stirring until the chemotherapeutic agent isdissolved; and c) extracting the solution of b) with saturated NaHCO₃and drying.

It will be understood by those of skill in the art that the methods formaking the compositions of the present invention can use any knownsolvents or mixtures thereof that will dissolve the chemotherapeuticagent. Moreover, other linkers can be used in the inventive methods toprepare the drug amphiphiles of the present invention. Known methods forextraction of the mixtures and drying can also be used.

These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

Example 1 Self-Assembly and Properties of a CPT-buSS-Tau DA Conjugate

To illustrate the feasibility of the present invention, a series ofcamptothecin (CPT) DA conjugates were synthesized by reaction of anactivated disulfide-functionalized CPT molecule with Tau protein-derivedpeptide that has 1, 2 or 4 cysteine residues (FIG. 1). These conjugateswere purified to homogeneity by reversed phase HPLC and their identityconfirmed by mass spectrometric methods.

The assembly of these conjugates into fibrous nanostructures wasconfirmed by dissolution into aqueous solution and subsequenttransmission electron microscopy (TEM) and cryo-TEM analysis (FIG. 2).Both mCPT-buSS-Tau (FIGS. 2a and 2b ) and dCPT-buSS-Tau (FIGS. 2c and 2d) were observed to form long nanofibers of widths 6.7±1 and 7.2±1.4 nm,respectively. Given these diameters are close to the expected molecularlengths (3.5 and 3.8 nm, respectively), it indicates these arecore-shell micellar structures with the CPT moiety sequestered at thecore. qCPT-buSS-Tau(FIG. 2e-g ) was observed to form filamentousstructures of 9.5 nm in width, with a dark centerline observedthroughout the nanostructures. This dark centreline is due to thedeposition of the negative staining agent, uranyl acetate, and suggeststhe structures possess a hollow core that may have collapsed during TEMsample preparation. The similarity of these structures to those of thetobacco mosaic virus and other structures in the literature suggest thatqCPT-buSS-Tau adopts a nanotube structure. This is further confirmed bythe observation of circular ends at the termini ends (FIG. 2g ). Thecomparable value of the nanotube wall thickness to the molecular length(4 nm vs. 4.1 nm) implies that the molecules are packed in a monolayeredrather than bilayered fashion. These results demonstrate that theincorporation of different numbers of CPT units into the drugamphiphiles can tune both the drug loading content and the assemblymorphologies.

The leading role of the Tau peptide in promoting the formation of1-dimensional nanostructures was confirmed by circular dichroism (CD)spectroscopy, with all conjugates showing the characteristic negativesignal of the beta sheet in the 220 nm region (FIG. 3a-c ).

The morphological differences between the conjugates can be ascribed tothe number of CPT molecules that each possesses. Both mCPT-buSS-Tau(FIG. 3a ) and dCPT-buSS-Tau (FIG. 3b ) show strong negative signals inthe CPT absorption region (at 250 nm and between 330 and 400 nm),clearly indicated they are packed in a chiral environment. These signalswere absent in DMSO (FIG. 4), where the conjugates are expected to existas single monomers. qCPT-buSS-Tau (FIG. 3c ) on the other hand displayedtwo bisignate CD signals at 265 and 366 nm and a strong positive signalat 389 nm. This bisignate Cotton effect is frequently observed inaggregated π-conjugated systems, resulting from excitonic couplingbetween two adjacent chromophores in a chiral orientation. The positivesign of the couplet signal at the higher wavelength suggests a positivechirality and a right-handed helical arrangement of the CPT moleculeswithin the nanotubes.

The critical micellization concentrations of mCPT-buSS-Tau,dCPT-buSS-Tau and qCPT-buSS-Tau were determined to be 207 nM, 74 nM and53 nM, respectively, by a fluorescence method (FIG. 5). These very lowvalues are in the range of macromolecular amphiphiles and are animportant property of these conjugates. A high value would lead to rapiddissociation of the nanostructure when diluted upon introduction intothe bloodstream, whereas a low value would impart greater stability tothe assembly during circulation.

Fluorescence analysis (FIG. 6) also shows that the CPT is present as theclosed lactone form with emission maxima around 433 nm, rather than theopen carboxylic acid (maxima around 446 nm). This is critical for it toexert a cytotoxic effect as the open carboxylic acid form is inactive.The combination of both conjugation to the hydroxyl and sequesteringwithin a hydrophobic core act to shift the equilibrium between theclosed and open forms towards the active closed (lactone) form.

The shielding of the degradable linker from the microenvironmentafforded by assembly into the nanostructures was confirmed by adegradation study that showed the conjugates were stable in 10 mMphosphate solutions at 37° C., at concentrations far above their CMCvalues (FIGS. 3d and 3e ). In the presence of the reducing agentglutathione, however, faster release was observed. The differences inrelease rates between the conjugates can be ascribed to their CMCvalues, with lower values resulting in a greater stability towards bothreduction and hydrolysis. These observations suggest a release mechanismas depicted in FIG. 3f , indicating that the self-assemblednanostructures can serve as reservoirs to provide a consistent supply ofCPT conjugate monomers that can be quickly converted to bioactive CPT inthe presence of GSH and maintain an effective intracellularconcentration.

In vitro efficacy of the synthesized DA conjugates towards a number ofcancer cell lines (FIGS. 7 and 8) was determined by sulforhodamine (SRB)assay, showing that these amphiphiles can exert a cytotoxic effect.

Methods for Example 1

All peptides were synthesized by a combination of automated and manualFmoc solid-phase synthesis techniques. Automated synthesis ofFmoc-GVQIVYKK-Rink (SEQ ID NO: 1) was performed using the Focus XCautomated peptide synthesizer (AAPPTEC, Louisville, Ky.) using theappropriate Fmoc amino acids (AAPPTEC) and HBTU/DIEA (4:3.98:6 relativeto the amine). Terminal cysteines and branching lysines were addedmanually as appropriate, using HATU instead of HBTU as the couplingagent in the same ratio as described above. 20% 4-methylpiperidine inDMF was used for Fmoc deprotections and 20% acetic anhydride in DMF wasused for N-terminal acetylation. Peptides were cleaved from the resinusing trifluoroaceticacid (TFA)/triisopropylsilane(TIS)/H₂O (95:2.5:2.5)for mCys-Tau and TFA/TIS/H₂O/ethane-dithiol (EDT) (90:5:2.5:2.5) fordCys-Tau and qCys-Tau (FIG. 9).

The syntheses of the CPT-linker derivatives CPT-buSS-Pyr and CPT-Malareshown in FIG. 10.

Synthesis of 4-(pyridin-2-yldisulfanyl)butanoic acid (buSS-Pyr).buSS-Pyr was prepared using a modified version of a previously reportedmethod. Briefly, 4-Bromobutyric acid (2.00 g, 12.0 mmol) and thiourea(1.06 g, 14.0 mmol) were refluxed in EtOH (50 ml) for 4 hours. NaOH(4.85 g, 121 mmol) in EtOH (50 ml) was added and reflux was continuedfor 16 hours. After cooling to room temperature, the solution wasfiltered and the filtrate concentrated in vacuo and dissolved in H₂O (50ml). Acidification to pH 5 with 4M HCl gave a cloudy solution that wasthen extracted with Et₂O. The organic extracts were dried over anhydrousNa₂SO₄ and concentrated to give 4-sulfanylbutyric acid as a clear oil(816 mg) that was used without further purification. 4-Sulfanylbutyricacid (816 mg, 6.68 mmol) was dissolved in MeOH (1 ml) and added dropwiseto a solution of 2-aldrithiol (3.03 g, 13.7 mmol) in MeOH (5 ml), whichdeveloped a yellow color, and was allowed to react overnight. Themixture was purified by reversed phase HPLC, collecting the major peak.The fractions were combined and lyophilized to give buSS-Pyr as a paleyellow viscous oil (1.02 mg, 37% over two steps). ¹H NMR (CDCl₃, 400MHz, 298K): δ_(H) (ppm) 8.59 (d, ³J_(HH)=4.6, 1H), 7.91-7.81 (m, 2H),7.30-7.25 (m, 1H), 2.88 (t, ³J_(HH)=7.1, 2H), 2.50 (t, ³J_(HH)=7.2, 2H),2.09-2.00 (m, 2H).

Synthesis of camptothecin-4-(pyridin-2-yldisulfanyl)butanoate(CPT-buSS-Pyr). CPT-buSSPyr was synthesized using a modified version ofa previously reported method. Camptothecin (200 mg, 0.574 mmol) wassuspended in DCM (32 ml) and dimethylaminopyridine (44 mg, 0.36 mmol),buSS-Pyr (208 mg, 1.22 mmol) and diisopropylcarbodiimide (436 μl, 3.51mmol) were added. The mixture was stirred until complete dissolution ofthe camptothecin had occurred (1.5 days), with TLC (3% MeOH in CHCl₃)showing complete consumption. The solution was then filtered, dilutedwith CHCl₃ (30 ml), extracted with sat. NaHCO₃ (50 ml), brine (50 ml),dried over Na₂SO₄ and concentrated in vacuo. The residue was purified byflash chromatography using EtOAc (1:1 500 ml) then 0.5% MeOH in EtOAc(250 ml). Product fractions were identified by TLC, combined and solventremoved in vacuo to give CPT-buSS-Pyr as a pale yellow solid (195 mg,61%); ¹H NMR (CDCl₃, 400 MHz, 298K): δ_(H) (ppm) 8.43 (d, 3J_(HH)=4.2,1H), 8.40 (s, 1H), 8.23 (d, ³J_(HH)=8.6, 1H), 7.94 (d, ³J_(HH)=8.2, 1H),7.84 (m, 1H), 7.70-7.65 (m, 2H), 7.60 (m, 1H), 7.20 (s, 1H), 7.04 (m,1H), 5.67 (d, ²J_(HH)=17.3, 1H), 5.40 (d, ²J_(HH)=17.2, 1H), 5.29 (s,2H), 2.86 (t, ³J_(HH)=7.1, 2H), 2.75-2.57 (m, 2H), 2.31-2.03 (m, 4H),0.97 (t, ³J_(HH)=7.5); ¹³C NMR (CDCl₃, 100 MHz, 298K): δ_(C) (ppm)172.1, 167.7, 160.3, 157.6, 152.6, 149.9, 149.1, 146.6, 146.1, 137.3,131.5, 131.4, 130.9, 129.9, 128.63, 128.62, 128.4, 128.3, 120.4, 96.1,76.2, 67.4, 50.2, 37.7, 32.4, 32.0, 31.2, 24.0, 7.9 (FIG. 11); MS(MALDI-TOF): 560.065 [M+H]⁺.

Synthesis of camptothecin-3-maleimido-propanoate (CPT-mal). CPT (50 mg,0.144 mmol) was suspended in DCM (8 ml) and DMAP (11 mg, 0.093 mmol),3-maleimido-propionic acid (70 mg, 0.288 mmol) and DIC (109 μl, 0.698mmol) were added. The mixture was stirred until complete dissolution ofCPT had occurred (overnight), with TLC (3% MeOH in CHCl₃) showingcomplete consumption. The solution was then diluted with CHCl₃ (30 ml),extracted with H₂O (20 ml), sat. NaHCO₃ (20 ml), brine (20 ml), driedover Na₂SO₄ and concentrated in vacuo. The residue was purified by flashchromatography using 3% MeOH in CHCl₃. Product fractions were identifiedby TLC, combined and solvent removed in vacuo to give CPT-mal as anoff-white solid (37 mg, 51%). NMR showed a maleimide impurity, but onlya single CPT compound—this was used without further purification; ¹H NMR(CDCl₃, 300 MHz, 298K): δ_(H) (ppm) 8.41 (s, 1H), 8.24 (d, ³J_(HH)=8.0,1H), 7.96 (d, 1H, ³J_(HH)=8.1, 1H), 7.85 (dd, ³J_(HH)=8.4, 1.5, 1H),7.68 (dd, ³J_(HH)=8.2, 1.2, 1H), 7.19 (s, 1H), 6.67 (s, 2H), 5.66 (d,²J_(HH)=17.2, 1H), 5.40 (d, ²J_(HH)=17.2, 1H), 5.30 (s, 2H), 3.95-3.75(m, 2H), 3.00-2.85 (m, 2H), 2.35-2.08 (m, 2H), 0.96 (t, ³J_(HH)=7.5,3H); ¹³C NMR (CDCl₃, 100 MHz, 298K): 170.6, 169.9, 167.6, 165.8, 162.8,157.4, 152.6, 149.2, 146.6, 145.8, 131.6, 131.0, 129.9, 128.6, 128.4,120.6, 96.3, 67.5, 50.3, 33.5, 32.6, 32.1, 22.8, 21.1, 7.9 (FIG. 12); MS(ESI): 499.9 [M+H]⁺.

The synthesis of the DA conjugates mCPT-buSS-Tau, dCPT-buSS-Tau andqCPT-buSS-Tau are shown in FIG. 13.

Synthesis of mCPT-buSS-Tau. mCys-Tau (14.6 mg, 13.5 μmol) was dissolvedin an N₂-purged DMSO solution CPT-buSS-Pyr (10 mg in 1.50 ml, 17.8 μmol)and shaken overnight. The reaction was diluted to 30 ml with 0.1%aqueous TFA, giving a slightly viscous solution that was then purifiedby reversed phase HPLC. Product fractions were combined and immediatelylyophilized. The pale-yellow solid obtained was dissolved in 25 mlnanopure water and the product concentration was determined by DTTcalibration to be 233 μM (8.9 mg, 43%). The solution was then aliquottedinto cryo-vials, lyophilized and stored at −30° C. HPLC purity >99%; MS(MALDI): 1526.78 [M+H]⁺ (FIG. 14).

Synthesis of dCPT-buSS-Tau. dCys-Tau (10.8 mg, 5.0 mmol) was dissolvedin an N₂-purged DMSO solution of CPT-buSS-Pyr (9 mg in 500 μl, 16.1μmol) and allowed to react for 3 days. The solution was diluted to 10 mlwith 0.1% aqueous TFA and purified by reversed phase HPLC. Productfractions were combined and immediately lyophilized. The pale yellowsolid obtained was dissolved in 15 ml nanopure water containing 0.08%TFA and 8% acetonitrile and the product concentration was determined byDTT calibration to be 72.9 μM (2.5 mg, 22%). The solution was thenaliquotted into cryo-vials, lyophilized and stored at −30° C. HPLCpurity >99%; MS (MALDI): 2248.039[M+H]⁺ (FIG. 15).

Synthesis of qCPT-buSS-Tau. qCys-Tau (3.5 mg, 1.91 μmol) was dissolvedin an N₂-purged DMSO solution of CPT-buSS-Pyr (10 mg in 500 μl, 17.8μmol) and allowed to react for 8 days. The solution was diluted to 10 mlwith 0.1% aqueous TFA and purified by reversed phase HPLC. Productfractions were combined and immediately lyophilized. The pale yellowsolid obtained was dissolved in 19.5 ml nanopure water containing 0.05%TFA and 25% acetonitrile and the product concentration was determined byDTT calibration to be 14.2 μM (1.0 mg, 15%). The solution was thenaliquotted into cryo-vials, lyophilized and stored at −30° C. HPLCpurity >99%; MS (MALDI): 3693.811[M+H]⁺ (FIG. 16).

Synthesis of mCPT-mal-Tau. The general scheme for synthesizing thecontrol molecules is shown in FIG. 17. mCys-Tau (5.6 mg, 5.2 μmol) wasdissolved in an N₂-purged DMSO solution of CPT-mal (2.6 mg in 250 μl,5.2 μmol) and shaken overnight. The reaction was diluted to 10 ml with0.1% aqueous TFA and purified by reversed phase HPLC. Product fractionswere combined and immediately lyophilized. The white solid obtained wasdissolved in 10 ml nanopure water and the product concentration wasdetermined by HPLC calibration to be 273 μM (4.3 mg, 53%). The solutionwas then aliquotted into cryo-vials, lyophilized and stored at −30° C.HPLC purity >99%; MS (MALDI): 1577.203[M+H]⁺ (FIG. 18).

C₈-Tau was synthesized by automated solid-phase synthesis ofFmoc-GVQIVYKK-Rink (SEQ ID NO: 1), followed by manual coupling of 4equivalents of octanoic acid using HBTU/DIEA in DMF. After cleavage withtrifluoroacetic acid/trisopropanol/water (95:2.5:2.5), the crude productwas precipitated with diethyl ether and purified by reversed phase HPLC.The product concentration was determined by UV-Vis analysis of thetyrosine absorption at 375 nm and aliquotted into cryo-vials,lyophilized and stored at −30° C. HPLC purity >99%; MS (MALDI): 1100.681(FIG. 19).

Example 2 Synthesis and Self-Assembly of a qCPT-buSS-Sup35 DA Conjugate

To illustrate the general applicability of the present invention wesynthesized an analogue of the qCPT-buSS-Tau DA using a β-sheet formingpeptide sequence derived from the yeast prion Sup35, CGNNQQNYKK (SEQ IDNO 5)—qCPT-buSS-Sup35.

Upon dissolution in aqueous solution, qCPT-buSS-Sup35 was found to formthe nanotube structure of similar dimensions as qCPT-buSS-Tau indicatingthat the replacement of one β-sheet forming peptide for another has noeffect on the structure that is adopted (FIG. 3h ).

CD analysis of the self-assembled structure shows the same pattern ofsignals as qCPT-buSS-Tau (FIG. 20), implying that the internal packingof the monomers is unaffected by the change in peptide sequence.

Methods for Example 2

qCys-Sup35 was synthesized in a similar manner to qCys-Tau (as describedin Example 1). Cleavage from the resin was affected by TFA/TIS/H₂O/EDT(90:5:2.5:2.5), and the crude peptide was purified by reversed phaseHPLC.

Synthesis of qCPT-buSS-Sup35. qCys-Sup35 (5.4 mg, 2.5 μmol) wasdissolved in an N₂-purged DMSO solution of CPT-buSS-Pyr (10.5 mg in 800μl, 18.8 μmol) and allowed to react for 5 days. The solution was dilutedto 10 ml with 0.1% aqueous TFA and purified by RP-HPLC. Productfractions were combined and immediately lyophilized. The pale yellowsolid obtained was dissolved in 4 ml nanopure water containing 0.1% TFAand the product concentration was determined by DTT calibration to be340 μM (2.0 mg, 22%). The solution was then aliquotted into cryo-vials,lyophilized and stored at −30° C. HPLC purity >99%; MS (ESI): 1925.97[M+2H]²⁺, 1291.94 [M+3H]³⁺ (FIG. 21).

Example 3 Synthesis and Self-Assembly of a Disulfanylcarbonate LinkedmCPT-etcSS-Tau Conjugate

To illustrate the applicability of the present invention to otherlinking moieties, a disulfanylcarbonate linked analogue, mCPT-etcSS-Tau(FIG. 22), of mCPT-buSS-Tau was synthesized by reaction of an activateddisulfide derivative of CPT—CPT-etcSS-Pyr (FIG. 23)—with thecysteine-functionalized Tau peptide, CGVQIVYKK (SEQ ID NO:1) (mCys-Tau).This carbonate-based linker can more effectively release the free CPTdrug upon reduction with glutathione, compared to thedisulfanylbutanoate linker (FIG. 24).

The self-assembly of mCPT-etcSS-Tau into nanostructures was confirmed bydissolution of the conjugate into aqueous solution and TEM analysisafter 24 hours incubation (FIG. 25a ). CD analysis confirmed thepresence of the expected □-sheet structure (FIG. 25b ).

Degradation studies of mCPT-etcSS-Tau (50 μM) in the presence of 10 mMglutathione at 37° C. indicated the fast effective release of unmodifiedCPT (FIG. 26a-b ) with no intermediary structures observed. In theabsence of glutathione, less than 15% degradation was observed over thesame 24 hour period.

A dose-response study illustrated the effect that the more efficientdrug release has on the cytotoxicity, with the carbonate-based linkerhaving a 3-fold increase in efficacy relative to the ester-baseddisulfanylbutanoate linker, and exhibiting similar toxicity to free CPT(FIG. 26c ).

This result demonstrates that alternative linkers can be incorporated tochange the release properties of the designed DA conjugates.

Methods for Example 3

Synthesis of 2-(pyridyl-disulfanyl)ethanol. The synthesis of thisprecursor was adapted from a previously reported procedure for theformation of activated disulfides. 2-Aldrithiol (1.29 g, 5.86 mmol) wasdissolved in MeOH (3.5 ml) and 2-mercaptoethanol (300 μl, 334 mg, 4.28mmol) was added dropwise over 5 min, the solution turning a yellowcolor. After 3 h, the solution was diluted with 0.1% aq. TFA (4.5 ml)and purified by RP-HPLC. Product fractions were combined and solventsremoved in vacuo. A solution of sat. NaHCO3 (15 ml) was added toneutralize the TFA, allowing to stand for 30 min before extracting intoDCM. The organic extract was dried over Na2SO4 and solvents removed togive 2-(pyridyl-disulfanyl)ethanol as pale yellow oil (561 mg, 70%). ¹H(300 MHz, CDCl₃, Me4Si) 2.91-2.99 (2H, m), 3.80 (2H, br s), 7.15 (1H,m), 7.41 (1H, dt, J1,3 8.0, 1.0), 7.54-7.63 (1H, m), 8.47-8.53 (1H, m).

Synthesis of Camptothecin-4-nitrophenyl carbonate. Camptothecin (100 mg,287 μmol) and nitrophenylchloroformate (203 mg, 1.00 mmole) weredissolved/suspended in dry DCM (15 ml) at 0° C. Dimethylaminopyridine(DMAP, 210 mg, 1.72 mmol) was added, turning the solution yellow. After3 h, the yellow-brown solution was filtered, washed with 1 N HCl (20ml), dried over Na₂SO₄ and concentrated in vacuo. Purification by flashchromatography—DCM (50 ml), 1:1 DCM/EtOAc (100 ml), EtOAc (200 ml), then1% MeOH in EtOAc (100 ml)—gave camptothecin-4-nitrophenyl carbonate as apale yellow solid (75 mg, 51%). ¹H (400 MHz, CDCl₃, Me₄Si) 1.01-1.12(3H, m) 2.16-2.45 (2H, m) 5.24-5.38 (2H, m) 5.42 (1H, d, J1,2 17.3) 5.72(1H, d, J1,2 17.2) 7.36-7.44 (3H, m) 7.70 (1H, ddd, J1,3 8.2, 6.9, 1.2)7.86 (1H, ddd, J1,3 8.5, 7.0, 1.5) 7.96 (1H, dd, J1,3 8.2, 1.3)8.17-8.28 (3H, m), 8.43 (1H, s).

Synthesis of Camptothecin-(4-pyridyldisulfanyl)ethyl carbonate(CPT-etcSS-Pyr). Camptothecin-4-nitrophenyl carbonate (70 mg, 136 μmol)and 2-(pyridyl-disulfanyl)ethanol (42 mg, 225 μmol) were dissolved indry DCM (15 ml), and DMAP (31 mg, 254 μmol) was added and the mixturewas refluxed (55° C.) overnight. After cooling, the mixture was washedwith 1 M NaHCO3 (3×15 ml) till colorless, dried over Na2SO4 andconcentrated in vacuo. Purification by flash chromatography-DCM (50 ml),1:1 DCM/EtOAc (300 ml), 1:3 DCM/EtOAc (100 ml), EtOAc (200 ml), 1% MeOHin EtOAc (100 ml), then 2% MeOH in EtOAc (100 ml)—to give CPT-etcSS-Pyras a pale yellow solid (57 mg, 75%). δ_(H) (400 MHz, CDCl3, Me4Si) 1.01(3H, t, J1,3 7.5), 2.10-2.21 (1H, m), 2.24-2.34 (1H, m), 3.06 (2H, t,J1,3 6.6), 4.30-4.42 (2H, m), 5.27-5.30 (2H, m), 5.39 (1H, d, J1,217.2), 5.69 (1H, d, J1,2 17.2), 7.03 (1H, td, J1,3 5.0, 3.4), 7.34 (1H,s), 7.62 (1H, d, J1,3 1.4), 7.63-7.64 (1H, m), 7.65-7.70 (1H, m), 7.83(1H, m), 7.94 (1H, dd, J1,3 8.2, 1.3), 8.22 (1H, d, J1,3 8.7), 8.39 (1H,s), 8.42 (1H, dt, J1,3 4.8, 1.4); ¹³C (100 MHz, CDCl3, Me4Si) 7.6, 31.9,36.9, 50.0, 66.4, 67.1, 78.0, 96.0, 119.9, 120.3, 120.9, 128.1, 128.2,128.4, 129.7, 130.7, 131.2, 137.2, 145.5, 146.5, 148.9, 149.7, 152.3,153.4, 157.3, 159.3, 167.3 (FIG. 27).

Synthesis of mCPT-etcSS-Pyr. mCys-Tau (22.8 mg, 21.2 μmol) was dissolvedin an N₂-purged DMSO solution of CPT-etcSS-Pyr (15.4 mg, 27.5 μmol) andallowed to react overnight. The solution was diluted to 9 ml with 0.1%aqueous TFA and purified by RP-HPLC. Product fractions were combined andimmediately lyophilized. The pale yellow solid obtained was dissolved in1:1 H2O/MeCN (10 ml) and the product concentration determined by DTTcalibration (S1.4) to be 1.61 mM (24.6 mg, 76%). The solution wasaliquotted into cryo-vials, lyophilized and stored at −30° C.; HPLCpurity >99%; MS (MALDI): 1528.990 (FIG. 28).

Example 4 Self-Assembly and Characterization of a PXL-buSS-Tau DAConjugate

To illustrate the applicability of the present invention to otherhydrophobic drugs, the paclitaxel analogue, PXL-buSS-Tau, ofmCPT-buSS-Tau was synthesized by reaction of an activated-disulfidederivative of PXL—PXL-buSSPyr—with the cysteine-functionalized Taupeptide, CGVQIVYKK (SEQ ID NO: 6) (mCys-Tau) (FIG. 29). The activateddisulfide buSSPyr linker was conjugated to PXL via the 2′-hydroxylgroup. It is known that modification of the C′2 hydroxyl position in PXLcan lead to loss of activity, so addition of the disulfide linker tothis position effectively creates a paclitaxel prodrug which can onlyexert its effect when taken into tumor cells and when the drug isreleased from the linker by glutathione.

The self-assembly of PXL-buSS-Tau into nanostructures was confirmed bydissolution of the conjugate into aqueous solution and TEM analysisafter 48 hours incubation. At 200 μM, PXL-buSS-Tau clearly formnanofibrous structures with diameters of 10-20 nm (FIG. 30), whilst at10 μM smaller fibers and spherical micellar structures are observed,suggestive of a higher CMC value when compared to the analogous CPTconjugate.

The CMC value of PXL-buSS-Tau was determined by encapsulation of thesolvatochromic dye Nile Red into the hydrophobic interior of thenanofiber structures. Fluorescence measurements of this fluorophore gavea CMC value in the range of 10 to 50 μM (FIG. 31a ).

In order to demonstrate different secondary structures at variousconcentrations below and above CMC value, circular dichroism spectrawere recorded at 5 μM and 100 μM (FIGS. 31b and 31c ). At concentrationslower below the CMC, PXL-buSS-Tau formed beta sheet secondary structures(negative absorption at 216 nm), with a positive absorption at 237 nmthat may arise from the n-π* transition of the PXL carbonyl groups at3C′ and 9C. At higher concentrations, single molecules aggregate tocylindrical nanofibers with paclitaxel packed inside as hydrophobiccore, leading to a significantly stronger signal at 237 nm.

PXL release studies were carried out at 100 μM and 5 μM concentrationsin PBS buffer at 37° C. (FIG. 32). Significant release of paclitaxel wasobserved for the 100 μM solution after 30 minutes in the presence ofglutathione, with the lower concentration of 5 μM showing a much fasterrelease than that of 100 μM—consistent with the DA conjugate existing asa greater proportion of monomers at this concentration below the CMC.After 2 hrs, around 70% of the PXL-buSS-Tau was cleaved by glutathioneat 5 μM while only 40% paclitaxel was released at 100 μM, againillustrating the protective effect that self-assembly intonanostructures has upon the biodegradable linker.

To evaluate anti-tumor efficacy, the cytotoxicity of PXL-buSS-Tautowards human breast cancer MCF-7, non-small cell lung cancer A549, andhuman prostate cancer PC-3 FLU cell lines was determined (FIG. 33).PXL-buSS-Tau exhibited a strong cytotoxic effect on all three cancercell lines, exhibiting similar efficacy to free paclitaxel. In addition,the cellular morphologies were observed to change, displaying a largersize that is consistent with G2-M arrest during the cell cycle.

Methods for Example 4

Synthesis of PXL-buSS-Pyr (C2′ isomer). Paclitaxel (185.6 mg, 0.22mmol), 4-(pyridin-2-yl-disulfanyl)butyric acid (100 mg, 0.44 mmol), DIC(68.36 μL, 0.44 mol), DMAP (26.7 mg, 0.22 mmol) were added to an ovendried flask under nitrogen and dissolved in anhydrous acetonitrile (12.7ml). The reaction was allowed to stir in the dark at room temperaturefor 48 hours. The solvents were removed in vacuo and the residue wasdissolved in chloroform and purified by flash chromatography (3:2EtOAc/hexanes), to give PXL-buSS-Pyr (0.108 g, 46.7%). HPLC purity >98%(FIG. 34); MS (ESI): m/z 1065.2 for [M+H]⁺ (FIG. 35).

Synthesis of PXL-buSS-Tau. Add AcCGVQIVYKK (SEQ ID NO: 6) (27.7 mg, 25.7μmol) and PXL-buSS-Pyr (54.7 mg, 51.4 μmol) into an oven dried flaskunder nitrogen and dissolved in anhydrous dimethylformamide (5 ml). Thereaction was allowed to stir for 16 hr after which the solution waspurified by reversed phase HPLC (30% to 95% acetonitrile in water with0.1% TFA over 45 minutes). Product-containing fractions were combinedand lyophilized to give PXL-buSS-Tau as a white powder (31.3 mg, 60%).HPLC purity >98% (FIG. 36); MS (ESI) 1031 [M+2H]²⁺ (FIG. 37).

Example 5 Self-Assembly and Characterization of a Dual Drug DA Conjugate

Incorporating two different drug molecules into a single self-assemblingentity presents a challenge when the overall properties of the conjugateare expected to depend strongly on the nature of those drugs. Forsimplicity, we used the reducible disulfylbutyrate linker to conjugateboth drugs to the hydrophilic peptide. However, in other embodiments,one can use linkers that would allow attachment via orthogonal reactionmechanisms. Such an approach opens up the ability of differential drugrelease through separate degradation pathways. The β-sheet formingpeptide was chosen to be a sequence derived from the Sup35 yeast prion,GN₂Q₂NYK₂ (SEQ ID NO: 7), with the two added lysine residues providing acharged head group and the glycine acting as a spacer. This sequence ismore hydrophilic than the Tau sequence we have previously utilized, andis expected to provide greater solubility to the final conjugate,CPT-PXL-Sup35. Conjugation of the two drugs is accomplished usingdirected disulfide formation, requiring the incorporation of twothiol-containing cysteine residues into the peptide. The total drugloading of this conjugate is fixed at 41%, with a CPT and PXL content of12 and 29%, respectively.

The dual DA, CPT-PXL-Sup35, was synthesized by statistical reaction witha 1:1 mixture of the activated disulfide drugs, CPT-buSS-Pyr andPXL-buSS-Pyr, in nitrogen purged DMSO (FIG. 38). Given the significantdifference in structure between the two drugs, CPT being predominantlyplanar, and PXL being bulky and three-dimensional, it was expected thatthere would be a subsequent difference in the product distribution,particularly with regard to the addition of the second drug. To probethis, dCys-Sup35 was reacted with one equivalent per thiol of the 1:1drug mixture, purifying the reaction by reversed-phase HPLC before ithad reached completion (FIG. 39 a). The resulting drug-containingspecies were isolated and calibrated in order to determine the absoluteamounts of each conjugate formed. It was found that the singly reactedspecies, CPT-Cys-Sup35 and PXL-Cys-Sup35, were formed in similarproportions (39 and 33% respectively), with each giving the two expectedpositional isomers, as indicated by the occurrence of two closelyseparated peaks for each species. These species were found to havereactive thiol groups, which upon dissolution in PBS were observed toform scrambled products through disulfide exchange (data not shown). Theremaining products of the reaction were found to be the hetero-dual DA,CPT-PXL-Sup35, and the two homo-dual DAs, dCPT-Sup35 and dPXL-Sup35. Theproduct distribution was observed to be biased towards the lesssterically crowded dCPT-Sup35, with only 6% each of the twoPXL-containing conjugates. Reaction of PXL-Cys-Sup35 with either of thetwo activated drugs may be hindered due to the bulky nature of PXL,potentially causing similar issues for the reaction of CPT-Cys-Sup35with PXL-buSS-Pyr. In order to push the reaction towards completion,dCys-Sup35 was allowed to react with a three-fold excess per thiol ofthe 1:1 activated drug mixture for 5 days (FIG. 39 b). The final productdistribution showed that the desired hetero-dual DA comprised 56% of themixture, with dCPT-Sup35 (31%) and dPXL-Sup35 (13%) making up theremainder. The low conversion to dPXL-Sup35 again clearly indicates thatthe bulky PXL causes steric hindrance during the directed disulfideexchange reaction.

Self-Assembly Characterization. Given that the bulky nature of PXLstrongly influenced the product distribution during synthesis, weinvestigated the effect it could have on the self-assembly of the dualDAs under aqueous conditions. Solutions of all three dual DAs wereprepared at 1 mM in water and allowed to age for 2 hours before dilutingto 100 μM. Transmission electron microscopy (TEM) imaging and circulardichroism (CD) spectra were recorded to evaluate the nanostructuresformed and how they evolved over time (FIG. 33). Initially, thehetero-dual DA, CPT-PXL-Sup35, was observed to form two types offilamentous nanostructures—small wormlike structures (7-8 nm widths),which display a strong tendency to curl up on themselves, andcomparatively longer twisted filaments ˜14 nm in width (FIGS. 40 a-b).If any β-sheet secondary structure is present, the CD analysis (FIG. 40f) indicates it is not predominant and is overwhelmed by the broadpositive signal at 230 nm that can be attributed to PXL n-π*transitions. The strong negative signal at 201 nm suggests that thepeptide may be adopting either a random coil or poly-prolinetype-II-like structure. Negative signals can also be observed for bothPXL and CPT π-π* transitions, though PXL generally exhibits strongcircular dichroism due to its three benzyl groups all being attached toasymmetric centers. CPT on the other hand, exhibits stronger CD when inan aggregated state, so the observation of a negative signal confirmsthat some degree of assembly is taking place.

After 24 hours or longer incubation time, TEM imaging shows that thetwisted filament morphology is the only nanostructure present andappears to have undergone significant growth to give contour lengths onthe order of several μm (FIGS. 40 c-d). CD analysis indicates that theelongation is coincident with β-sheet formation, exhibiting the typicalsignal at 218 nm. The negative signals for the PXL and CPT π-π*transitions also undergo a blue-shift, suggesting a change in theirsurrounding environment. At the widest point, these fibrils are 13.9±1.7nm across, and at their narrowest appear to be approximately half ofthis value. These observations are thus suggestive of two entwinedfilaments, each of which is approximately 7 nm in width. Further agingdoes not appear to lead to any significant changes in the nanostructure,as indicated by TEM and CD analysis. Cryo-TEM imaging, a technique thatpreserves the solution state structure in vitreous ice, confirms thatlong fibrous structures are obtained (FIG. 40 e). Due to practicallimitations, however, the capture of high magnification images was notpossible and consequently the twisted nature could not be verified usingthis technique.

The presence and morphologies of the two types of nanostructure in theinitial stages bears remarkable similarity to the formation of amyloidfibrils and filaments formed by peptidomimetics. It has been proposedthat amyloid fibrils rich in β-sheets assemble via a series ofintermediate structures, beginning with narrow filaments that can twistaround one another to give fibrils comprised of two or more filaments orundergo lateral associations to give a non-twisted ribbon-like assembly.Further changes can then occur to give twisted ribbons and tubes. In thepresent invention, we observe the initial formation of short filamentsthat associate with one another to give two-filament fibrils. It isthought that the short filaments are kinetically-favorable structuresthat result from the rapid hydrophobic collapse of CPT-PXL-Sup35molecules upon dissolution in water. The absence of the characteristicβ-sheet absorption in the CD spectrum at early assembly stages indicatesthe elongation may not be directly linked to hydrogen bonding amongSup35 peptides, but rather as a result of molecular pacing associatedwith the hydrophobic segments. It is very likely that a β-sheet issterically hindered due to the mismatch in size between CPT and PXL.Given time, however, it appears that two of these filaments cometogether and by entwining can undergo reorganization of their internalstructure, forming β-sheets that promote elongation of the fibrils togive the extended structures observed at later time points. Furthermore,a small bisignate peak centered at 378 nm (one of the CPT π-π*transitions) in the CD spectrum hints at some potential CPT-CPTelectronic interactions that may also play a role in the assemblyprocess. Also, it is important to point out that, unlike the structuralpolymorphism shown by amyloid fibrils, the data show that thetwo-filament fibrils are the end-point in the structural evolution, asno ribbon-like or other structures could be seen at any time point. Itis thought that this may be a combination of the hydrophobic domain ofCPT-PXL-Sup35 being shielded from the aqueous environment (theamphiphilic nature of the designed Das of the present invention) and thefibril's twisted nature, with both properties preventing any furtherlateral associations that would give a ribbon-like morphology.

Further evidence for the evolution of the nanostructure from smallfilaments to twisted two-filament fibrils is provided by studying theeffect of high salt concentration on the morphology. Dilution of the 1mM stock solution of CPT-PXL-Sup35 that had been aged for 2 hours to 100μM in Dulbecco's phosphate-buffered saline (1×DPBS) was seen toeffectively retard the structural evolution, giving only the smallfilament morphology (FIG. 41 a). Very little shift toward the twistednanofilament structure was observed over the course of several days,with TEM analysis indicating only the smaller nanostructure withoccasional examples of the entwined morphology (FIG. 41 b). CD analysisconfirms that there is a slow shift toward the β-sheet structure, thoughit does not reach the extent of the sample aged in water even after 3days (FIG. 42 a). This slower rate of structural evolution may be due toincreased shielding/crosslinking of the protonated lysine residues bythe multivalent phosphate anions. Reorganization of the internalstructure requires that the assembly be dynamic in nature, the extent ofwhich would be greater in the absence of the phosphate ions. Byproviding a screening effect, the phosphate anions significantly reducethe rate at which reorganization can occur. In contrast, dilution into1×DPBS of a 1 mM sample (prepared in water) that had been allowed to agefor 8 days exhibited the same twisted fibril morphology observed in purewater (FIG. 41 c) and possessed a CD spectrum consistent with that inpure water (FIG. 42 b). This indicates that dilution into DPBS does notdisrupt the nanostructures already present in solution and that themorphologies observed are true representations of the assemblies presentin the stock solution. An interesting point to note is that interfibrilbundling is not observed despite the increased charge shielding affordedby the phosphate ions, with the fibrils being observed only as singleelements. This suggests that their twisted nature does indeed reduce thelikelihood of further lateral associations.

The observations from this self-assembly study clearly demonstrate theeffect that the PXL molecule can have when incorporated into a drugamphiphile. In its absence, the planar CPT can easily adopt afilamentous morphology (FIG. 43 a), but the replacement of one CPT bythe bulkier PXL results in a slower assembly process that ultimatelygives a twisted fibrillar nanostructure. Replacement of both CPTs withPXL on the other hand leads to the formation of only small micellarstructures (FIG. 43 b).

Nanostructure Stability. To gain more information on the stability ofthese nanostructures, a critical aggregation constant (CAC) study wasperformed based on encapsulation of the solvatochromic fluorophore, NileRed. The emission spectrum of this dye differs significantly when placedin hydrophobic or hydrophilic environments and thus can be used as aprobe for assembly processes. Accordingly, various concentrations ofCPT-PXL-Sup35 were incubated overnight with 1 μM Nile Red beforerecording the emission spectra (exciting at 550 nm). Plotting the 640 nmemission data against the conjugate concentration gave a CAC valuebetween 20-30 μM for CPT-PXL-Sup35 (FIG. 44). This value fits into theCMC range reported by the Tirrell group for peptide amphiphile systems(Biochemistry 2009, 48, 3304-3314). A similar study for dCPT-Sup35,however, failed to give a satisfactory response as little increasedfluorescence was observed, even at concentrations known to formsignificant nanostructures—likely due to poor penetration of the dyeinto the assembly that is expected to have a high degree of internalorder arising from the π-π stacking of the CPT units. CD analysis of thetwo conjugates at 5 μM, recorded immediately after dilution of a 100 μMsolution, revealed that disassembly of the nanostructures occurs at thisconcentration, with little or no β-sheet structure present. It should benoted that the Tau peptide analogue of dCPT-Sup35 in our previous workdid not show such behavior, exhibiting the typical β-sheet signal evenwhen diluted below 1 μM. This highlights the importance of the peptidesequence in the overall structural stability of drug amphiphiles. Thisdifference in CAC values might due to the fact that the Sup35 peptideGNNQQNY (SEQ ID NO: 8) contains more polar amino acids than the Taupeptide VQIVYK (SEQ ID NO: 9), potentially affecting their hydrogenbonding capacity between themselves and with water.

Drug Release. In order to evaluate the ability of CPT-PXL-Sup35 torelease its therapeutic cargo, we incubated a 50 μM solution of thisdual DA at 37° C. in the presence or absence of the cancer-relevantreducing agent, glutathione (GSH) (FIG. 45 a). Aliquots were taken atvarious time points, quenching the reaction by the addition of 1 M HCland flash freezing in liquid nitrogen. These samples were then analyzedby HPLC to determine the concentration of the important reactioncomponents at each time point (CPT-PXL-Sup35, CPT and PXL). Theconjugate was observed to degrade rapidly in the presence of GSH, beingcompletely consumed within 2 hours, whereas >80% remained after 8 hoursincubation in its absence. It can be seen that, initially at least, PXLis released almost twice as fast as CPT in the presence of GSH, perhapsdue to the PXL-ester bond being more labile than the CPT-esterbond—hydrolysis of a 2° alcohol ester (PXL) is expected to be fasterthan that of a 3° alcohol ester (CPT) due to steric considerations ofthe tetrahedral intermediate formed. The increase in the free drugconcentration continues beyond the 2 hour time that it takes tocompletely degrade the conjugate as the linker-modified form of thedrugs, CPT-buSH and PXL-buSH, are released first, before undergoingfurther hydrolysis to give the free drugs. Homo- and hetero-disulfideproducts, such as (CPT-buS)₂, (PXL-buS)₂, and CPT-buS-Sbu-PXL, can alsobe observed before they too undergo reduction and/or hydrolysis (FIG. 45b). Formation of these homo- and hetero-disulfide degradation productsare likely a result of the supramolecular nature of the DAnanostructures, which leads to a high local concentration of releasedthiol products.

Cytotoxicity Study. The activity of the synthesized conjugates toprohibit proliferative ability was assessed through the determination ofa dose-response relationship against PXL-sensitive and -resistantcervical cancer cell lines—KB-3-1 and KB-V1, respectively. Both celllines were individually incubated for 48 hours with the conjugates aloneor in combination, either individually to determine their sensitivity tothe conjugates (FIGS. 46 a-b) and also as a co-culture to mimic aheterogeneous tumor (FIG. 46 c). As expected, the PXL-sensitive KB-3-1cells (FIG. 46 a) display nano-molar sensitivity to the PXL-containingconjugates, dPXL-Sup35 (alone or in combination with dCPT-Sup35) andCPT-PXL-Sup35 and moderate sensitivity towards dCPT-Sup35, consistentwith the respective activities of free PXL and CPT. At higherconcentrations, the experiments that combined CPT and PXL, either asfree drugs or drug amphiphiles, all showed a reduction in theanti-proliferative activity to levels similar to free CPT. Thisantagonistic behavior has been previously observed, and is suspected toarise from an up-regulation of anti-apoptosis genes on co-treatment withCPT that would inhibit the mechanism by which PXL exerts itscytotoxicity. The PXL-resistant KB-V1 cells (FIG. 46 b) show moderatesensitivity toward all the CPT-containing drug conjugates, with littledifference compared to free CPT, whereas PXL and dPXLSup35 both show noactivity.

Against a co-culture of KB-3-1 and KB-V1 cervical cancer cells, whichserves to better mimic a heterogeneous tumor that consists of more thanone phenotype, a similar trend in behavior is observed (FIG. 46 c).Cells treated with PXL-species only (free PXL or dPXL-Sup35) show nogreater than 50% loss in viability after 48 hours, as only thePXL-sensitive KB-3-1 cells will be affected. Those treated withCPT-species only (free CPT or dCPT-Sup35) display moderate activity,similar to that observed for each individual cell line. Incubation withthe homo-dual DA, CPT-PXL-Sup35, or a combination of the two hetero-dualDAs, dCPT-Sup35 and dPXL-Sup35, appears to effectively kill the KB-3-1cells at lower concentrations, with only the KB-V1 cells likely tosurvive. The antagonistic behavior is again observed at higherconcentrations and results in a comparable effect to free CPT as before.Surprisingly, the combination of free CPT and PXL exhibited a greaterthan expected anti-proliferative effect, with only 30% of cellsremaining when treated with a 10 nM concentration of each drug. Thisimplies that around 20% of the PXL-resistant KB-V1 cells are alsoaffected despite having little sensitivity to CPT at this concentration,indicating that the observed cytotoxicity is due to PXL. While theantagonistic effect is still observed at higher concentrations, theoverall cytotoxicity remains greater than CPT alone. Given that thiseffect is not seen for the KB-V1 mono-culture, it suggests that the useof CPT can perhaps, to some degree, sensitize PXL-resistant cells to PXLwhen co-cultured with sensitive cells.

We have demonstrated that the incorporation of two structurally distinctanticancer drug molecules into a single amphiphilic entity is asuccessful strategy for the creation of well-defined nanostructures.Combining CPT and PXL into a single hetero-dual drug amphiphile wasfound to give nanostructures that possess a two-filament fibrilmorphology in which two narrower filaments entwine about one another.These results illustrate that the conjugation of two drugs withdiffering packing preferences onto one conjugate can be accomplishedwithout compromising the self-assembly or chemotherapeutic properties.This opens up the ability of simultaneous delivery of two drugs to thesame location at the same time with the potential for a great degree ofcontrol.

Methods for Example 5

Peptide synthesis (Ac-Cys)₂KGN₂Q₂NYK₂-NH₂ (dCys-Sup35) (SEQ ID NO: 10)was synthesized using a combination of automated (Focus XC automatedpeptide synthesizer, AAPPTEC, Louisville, Ky., USA) and manual solidphase synthesis techniques, employing standard Fmoc chemistry protocols.Fmoc deprotections were performed using 20% 4-methylpiperidine in DMFand couplings were carried out using aminoacid/O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU)/DIEA (4:3.98:6 relative to the resin) in DMF(with 2 minute activation time). Acetylation was carried out manuallyusing 20% acetic anhydride in DMF after N-terminal Fmoc deprotection.The branching lysine and terminal cysteines were introduced manuallyusing Fmoc-Lys(Fmoc)-OH and0-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU).

Dual Drug Amphiphile Synthesis. dCys-Sup35 (21 mg, 14 μmol) wasdissolved in a solution of an N₂-purged DMSO containing a 1:1 mixture ofCPT-buSS-Pyr (12 mg, 21 μmol) and PXL-buSS-Pyr (22.5 mg, 21 μmol) andallowed to react for 5 days. Analytical HPLC showed the reaction wasvirtually complete, giving the expected doubly reactedproducts—dCPTSup35, dPXL-Sup35 and CPT-PXL-Sup35. The reaction mixturewas diluted to 10 mL with 0.1% TFA in acetonitrile/water (2:3) andpurified by preparative RP-HPLC. The appropriate product fractions werecombined and lyophilized. The powders obtained were then re-dissolved,calibrated and aliquotted into cryo-vials before re-lyophilization asdescribed below.

The isolated dCPT-Sup35 was dissolved in 3 mL H2O. Calibration based onthe CPT absorbance gave a conjugate concentration of 448 μM. Yield=3.2mg, 10%. ESI-MS (%): 1226.3 (100) [M+2Na]²⁺, 1204.2 (69) [M+2H]²⁺,1215.3 (49) [M+H+Na]²⁺, 810.4 (26) [M+2H+Na]³⁺ (FIG. 47).

The isolated CPT-PXL-Sup35 was dissolved in 6 mL of 1:1 MeCN/H₂O.Calibration based on the CPT absorbance gave a conjugate concentrationof 400 μM. Yield=7.0 mg, 17%. ESI-MS (%): 1457.0 (100) [M+2H]²⁺, 1468.4(41) [M+H+Na]²⁺, 1478.9 (31) [M+2Na]²⁺ (FIG. 48).

The isolated dPXL-Sup35 was dissolved in 3 mL of 1:2 MeCN/H₂O.Calibration based on the PXL absorbance gave a conjugate concentrationof 204 μM. Yield=2.1 mg, 4%. ESI-MS (%): 1710.6 (100) [M+2H]²⁺, 1721.5(8) [M+H+Na]²⁺, 1147.9 (6) [M+2H+Na]³⁺, 1732.3 (2) [M+2Na]²⁺ (FIG. 49).

Transmission Electron Microscopy. Samples were prepared by depositing 7μL of the appropriate solution onto a carbon-coated copper grid(Electron Microscopy Services, Hatfield, Pa., USA), wicking away theexcess solution with a small piece of filter paper. Next, 7 μL of a 2 wt% aqueous uranyl acetate solution was deposited and the excess solutionwas carefully removed as above to leave a very thin layer. The samplegrid was then allowed to dry at room temperature prior to imaging.Bright-field TEM imaging was performed on a FEI Tecnai 12 TWINTransmission Electron Microscope operated at an acceleration voltage of100 kV. All TEM images were recorded by a SIS Megaview III wideangle CCDcamera.

Cryogenic Transmission Electron Microscopy. 6 μL of sample solution wasplaced on a holey carbon film supported on a TEM copper grid (ElectronMicroscopy Services, Hatfield, Pa., USA). All the TEM grids used forcryo-TEM imaging were treated with plasma air to render the lacey carbonfilm hydrophilic. A thin film of the sample solution was produced usingthe Vitrobot with a controlled humidity chamber (FEI). After loading ofthe sample solution, the lacey carbon grid was blotted using presetparameters and plunged instantly into a liquid ethane reservoirpre-cooled by liquid nitrogen. The vitrified samples were thentransferred to a cryo-holder and cryo-transfer stage that was cooled byliquid nitrogen. Imaging was performed using a FEI Tecnai 12 TWINTransmission Electron Microscope (100 kV) and images were recorded by a16 bit 2K×2K FEI Eagle bottom mount camera. To prevent sublimation ofvitreous water, the cryo-holder temperature was maintained below −170°C. during the imaging process.

Circular Dichroism. CD spectra were recorded on a Jasco J-710spectropolarimeter (JASCO, Easton, Md., USA) using a 1 mm path lengthquartz UV-Vis absorption cell (Thermo Fisher Scientific, Pittsburgh,Pa., USA). Background spectra of the solvents were acquired andsubtracted from the sample spectra. Collected data was normalized withrespect to sample concentration and β-sheet forming residues.

Drug Release Protocol. Briefly, a 100 μM solution CPT-PXLSup35 indeionized water was freshly prepared before the experiment and dilutedto 50 μM with sodium phosphate buffer (pH 7.4, 20 mM) with or withoutGSH (20 mM). The solutions were incubated at 37° C. and sampled at 0,0.17, 0.5, 1, 2, 4, 6, and 8 hours. The samples were acidified by theaddition of 0.2 μL of 2 M HCl, flash frozen with liquid nitrogen andstored at −30° C. until analysis by RP-HPLC was performed. Thedegradation of CPT-PXL-Sup35 was monitored by RP-HPLC using thefollowing conditions: 237 nm detection wavelength; 1 ml/min flow rate;mobile phase was 0.1% aqueous TFA (A) and acetonitrile containing 0.1%TFA; gradient is given in Table 1.

TABLE 1 HPLC gradient used for drug release study. Time (min) MobilePhase A (%) Mobile Phase B (%) 0 65 35 5 65 35 18 13 87 21 13 87 22 6535 25 65 35

The concentrations of CPT-PXL-Sup35, CPT and PXL were determined bymeasuring the area of the respective peaks in the HPLC chromatogram andcomparing against a calibration curve for each species.

Cell Culture. KB-3-1 and KB-V1 ovarian cancer cells were cultured inDMEM (Invitrogen) containing 10% fetal bovine serum (FBS, Invitrogen)and 1% of antibiotics (Invitrogen), and 1 μg/mL vinblastine was addedfor KB-V1 to maintain its multidrug resistance. The two cell types wereincubated at 37° C. in an Oasis humidified incubator with a 5% CO₂atmosphere (Caron, Marietta, Ohio).

Cytotoxicity Protocol. KB-3-1, KB-V1 or their co-culture (1:1) wereseeded onto 96-well plate (5×10³ cells/well) and allowed to attachovernight. PXL-CPT-Sup35 was diluted with fresh medium and incubatedwith cells immediately to achieve final conjugate concentrations of0.01, 0.1, 1, 10, 100 and 1000 nM. Medium containing the sameconcentration of PXL or/and CPT in the form of free drugs or conjugates(dCPT-Sup35 or dPXL-Sup35) were also used to incubate the cells, withnon-treated cells (solvent only) as the control group. After 48 hoursincubation, the cell viability was determined using the SRB methodaccording to the manufacturer's protocol (TOX-6, Sigma, St. Louis, Mo.).

Example 6 Preparation of a αv-β3 Integrin-Targeted DA, mCPT-buSS-Tau-RGD

An αv-β3 integrin-targeted DA, mCPT-buSS-Tau-RGD, was synthesized byreaction of the activated-disulfide, CPT-buSS-Pyr, with thecysteine-functionalized peptide, CGVQIVYKKGRDG (SEQ ID NO: 12) in DMSO.The peptide was synthesized using standard solid-phase Fmoc peptidesynthesis techniques and purified by RP-HPLC prior to conjugation withthe activated disulfide. The DA was isolated by RP-HPLC.

A folate-targeting DA was synthesized by reaction of theactivated-disulfide, PXL-buSS-Pyr, with the cysteine-functionalizedpeptide, CGNNQQNYKKGK (folate) (SEQ ID NO: 11) in DMSO. Thefolate-functionalized peptide was synthesized by 1) synthesis of theprotected peptide using solid-phase Fmoc synthesis techniques, employingan Mtt-protected lysine at the C-terminal to allow selectivefunctionalization of this residue; 2) removal of the Mtt protectinggroup using a 4% TFA solution in DCM with 5% triisopropylsilane; 3)conjugation of a suitably-protected folate was performed using HBTU andDIEA as coupling reagents, to give the folate-peptide; 4) cleavage anddeprotection was performed using standard protocols, followed by RP-HPLCpurification.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A composition comprising: D-L-PEP; wherein D is 1 or more hydrophobic drug molecules; L is 1 or more biodegradable linkers; and PEP is a hydrophilic peptide.
 2. (canceled)
 3. The composition of claim 1, wherein the linker L is capable of being cleaved intracellularly by glutathione.
 4. (canceled)
 5. The composition of claim 2, wherein PEP is selected from the following peptide sequences: NNQQNY (SEQ ID NO: 2); GRKKRRQRRRPPQ (SEQ ID NO: 3); LLKKLLKLLKKLLK (SEQ ID NO: 4); CGNNQQNYKK (SEQ ID NO 5); GNNQQNYKK (SEQ ID NO: 7); GNNQQNY (SEQ ID NO: 8); and CCKGNNQQNYKK (SEQ ID NO: 10) and derivatives thereof, wherein the derivatives comprise 1 to 10 additional amino acids on either the N-terminal or C-terminal end of PEP.
 6. The composition of claim 1, wherein D represents 2 hydrophobic drug molecules, or 3 hydrophobic drug molecules, or 4 hydrophobic drug molecules.
 7. The composition of claim 1, wherein D represents 1 to 4 hydrophobic drug molecules which can be the same or different.
 8. The composition of claim 1, wherein the drug molecule is selected from the group consisting of paclitaxel, camptothecin, anthracyclines, carboplatin, cisplatin, daunorubicin, doxorubicin, methotrexate, vinblastine, and vincristine.
 9. The composition of claim 1, wherein the linker is disulfanylbutanoate.
 10. The composition of claim 1, further comprising a pharmaceutically acceptable carrier.
 11. The composition of claim 10, further comprising at least one additional biologically active agent.
 12. A composition comprising 1 to 4 hydrophobic chemotherapeutic molecules linked via 1 to 4 disulfanylbutanoate linking molecules to the peptide CGVQIVYKK (SEQ ID NO: 6).
 13. The composition of claim 12, wherein the chemotherapeutic molecule is paclitaxel and/or camptothecin.
 14. The composition of claim 13, further comprising a pharmaceutically acceptable carrier.
 15. The composition of claim 14, further comprising at least one additional biologically active agent.
 16. A composition comprising: D-L-PEP-T; wherein D is 1 or more hydrophobic drug molecules; L is 1 or more biodegradable linkers; PEP is a hydrophilic peptide; and T is a targeting ligand.
 17. (canceled)
 18. The composition of claim 16, wherein the linker L is capable of being cleaved intracellularly by glutathione.
 19. (canceled)
 20. The composition of claim 16, wherein PEP is selected from the following peptide sequences: NNQQNY (SEQ ID NO: 2); GRKKRRQRRRPPQ (SEQ ID NO: 3); LLKKLLKLLKKLLK (SEQ ID NO: 4); CGNNQQNYKK (SEQ ID NO 5); GNNQQNYKK (SEQ ID NO: 7); GNNQQNY (SEQ ID NO: 8); and CCKGNNQQNYKK (SEQ ID NO: 10), CGVQIVYKKGRDG (SEQ ID NO: 11), and derivatives thereof, wherein the derivatives comprise 1 to 10 additional amino acids on either the N-terminal or C-terminal end of PEP.
 21. The composition of claim 16, wherein D represents 2 hydrophobic drug molecules, or 3 hydrophobic drug molecules, or 4 hydrophobic drug molecules.
 22. The composition of claim 16, wherein D represents 1 to 4 hydrophobic drug molecules which can be the same or different.
 23. The composition of claim 16, wherein T is selected from the group consisting of RGD, RGDS (SEQ ID NO: 13) peptide, folate and methotrexate. 